Levetiracetam Immunoassays

ABSTRACT

Methods, compositions and kits are disclosed directed at levetiracetam derivatives, immunogens, signal generating moieties, antibodies that bind levetiracetam and immunoassays for detection of levetiracetam.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 61/108,369 filed Oct. 24, 2008, whichis incorporated herein by reference in its entirety and for allpurposes.

BACKGROUND

The chemical name of levetiracetam, a single enantiomer, is(−)-(S)-α-ethyl-2-oxo-1-pyrrolidine acetamide, its molecular formula isC₈H₁₄N₂O₂ and its molecular weight is 170.21.

Levetiracetam is chemically unrelated to existing antiepileptic drugs(AEDs). It has the following structural formula:

Situations in which AED therapeutic drug monitoring (“TDM”) are mostlikely to be of benefit have been described (Epilepsia, 49(7):1239-1276,2008). They include (1) when a person has attained the desired clinicaloutcome, to establish an individual therapeutic concentration which canbe used at subsequent times to assess potential causes for a change indrug response; (2) as an aid in the diagnosis of clinical toxicity; (3)to assess compliance, particularly in patients with uncontrolledseizures or breakthrough seizures; (4) to guide dosage adjustment insituations associated with increased pharmacokinetic variability (e.g.,children, the elderly, patients with associated diseases, drugformulation changes); (5) when a potentially important pharmacokineticchange is anticipated (e.g., in pregnancy, or when an interacting drugis added or removed); and (6) to guide dose adjustments for AEDs withdose-dependent pharmacokinetics.

Therapeutic drug management of levetiracetam would serve as an excellenttool to ensure compliance in administering chemotherapy with the actualprescribed dosage and achievement of the effective serum concentrationlevels. The role of TDM for levetiracetam may also be useful in managingpatients that are overdosed.

To date no anti-levetiracetam antibody has been produced and nocommercially available immunoassay has been developed for levetiracetam.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows chemical structures: (1) levetiracetam, (2)2-pyrrolidone-N-butyric acid metabolite.

FIGS. 2a and 2b are flow diagrams illustrating embodiments of a methodfor performing an immunodiagnostic assay for levetiracetam. The rate ofincreasing absorbance at 340 nm due to the conversion of NAD⁺(Nicotinamide adenine dinucleotide reduced) to NADH (Nicotinamideadenine dinucleotide oxidized) is related to the concentration oflevetiracetam in the sample by a mathematical function. The enzymereaction is catalyzed by levetiracetam-G6PDH (Glucose-6-phosphatedehydrogenase) conjugate.

FIG. 3 shows a typical calibration curve for a competitive homogeneousimmunoassay for levetiracetam in pooled human serum calibrator matrixusing a Roche Hitachi 917 analyzer.

FIG. 4 shows a mass spectrograph of Compound 44.

FIG. 5 shows a ¹H NMR spectrum of Compound 44.

SUMMARY

Methods, compositions and kits are disclosed directed at levetiracetamderivatives, immunogens, signal generating moieties, antibodies thatbind levetiracetam and immunoassays for detection of levetiracetam.

The embodiments provide for detection of levetiracetam in a sample. Avariety of haptens, hapten-reactive partner conjugates, haptenderivatives, receptors, methods, and kits are useful in thisdetermination.

A certain embodiment is a compound of Formula 1 shown below:

wherein R₁ is —Y—Z, and Y is a linking group and Z is selected from thegroup consisting of hydrogen, —OH, —SH, —S-acyl; —O-lower alkyl,halogen, NH₂, -epoxy, -maleimidyl, haloacetamide, carboxyl and all itsactivated forms including hydroxysuccinimidyl, -succinimidyl,-carbonate, anhydride, imidate, an immunogenic carrier, a protein, and alabel, and including acid salts thereof.

A certain embodiment is a compound of Formula 2 shown below:

wherein R₂ is —Y—Z, and Y is a linking group and Z is selected from thegroup consisting of hydrogen, —OH, —SH, —S-acyl; —O-lower alkyl,halogen, NH₂, -epoxy, -maleimidyl, haloacetamide, carboxyl and all itsactivated forms including hydroxysuccinimidyl, -succinimidyl,-carbonate, anhydride, imidate, an immunogenic carrier, a protein, and alabel, and including acid salts thereof.

A certain embodiment is a compound of Formula 3 shown below:

wherein R₃ is —Y—Z, and Y is a linking group and Z is selected from thegroup consisting of hydrogen, —OH, —SH, —S-acyl; —O-lower alkyl,halogen, NH₂, -epoxy, -maleimidyl, haloacetamide, carboxyl and all itsactivated forms including hydroxysuccinimidyl, -succinimidyl,-carbonate, anhydride, imidate, an immunogenic carrier, a protein, and alabel, and including acid salts thereof.

The disclosure provides a method for determining a presence oflevetiracetam. The method comprises providing in combination in amedium: (a) a sample suspected of containing the compound; and, (b) anantibody raised against a compound of any of the above formulas 1-3. Themedium is examined for the presence a complex comprising the compoundand the antibody where the presence of such as complex indicates thepresence of the compound in the sample. In one aspect of the aboveembodiment, the combination further comprises a labeled conjugate of theabove compound.

A certain embodiment is a kit for determining a presence oflevetiracetam. The kit comprises (a) an antibody reactive to a commonepitope present in levetiracetam and compounds shown in formulas 1-3;(b) ancillary reagents for determining the compound; and, (c) a labeledconjugate of a compound of any of the above formulas 1-3. The antibodyof the kit may be an antibody raised against a compound of any of theformulas 1-3 above.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms aredefined for purposes of the present disclosure.

Analyte The compound or composition to be measured, the material ofinterest such as levetiracetam.

Sample Suspected of Containing Analyte

Any sample which is reasonably suspected of containing analyte can beanalyzed by the method of the embodiments. Such samples can includehuman, animal or man-made samples. The sample can be prepared in anyconvenient medium which does not interfere with the assay.

Typically, the sample is an aqueous solution or a natural fluid,preferably, urine, whole blood, serum, plasma, cerebral-spinal fluid, orsaliva more preferably, serum.

Measuring the Amount of Analyte

Quantitative, semiquantitative, and qualitative methods as well as allother methods for determining analyte are considered to be methods ofmeasuring the amount of analyte. For example, a method which merelydetects the presence or absence of analyte in a sample suspected ofcontaining an analyte is considered to be included within the scope ofthe embodiments. Synonyms for the phrase “measuring the amount ofanalyte” which are contemplated within the scope of the embodimentsinclude, but are not limited to, detecting, measuring, or determininganalyte; detecting, measuring, or determining the presence of analyte;and detecting, or determining the amount of analyte.

Human Serum

“Human serum”, as used herein, refers to the aqueous portion of humanblood remaining after the fibrin and suspended material (such as cells)have been removed.

Immunoassay

As used herein, the terms “immunoassay” or “immunodiagnostic” refer tolaboratory techniques or test systems that make use of the bindingbetween an antigen or analyte and an antibody in order to identifyand/or quantify at least one of the specific antigen or analyte orspecific antibody in a biological sample.

As used here, the term “competitive immunoassay” refers to aexperimental protocol in which a known amount of an identifiable antigenor analyte competes with another antigen or analyte for binding with anantibody. That is, a known antigen or analyte that binds with a knownantibody is combined with a sample that is suspected of containinganother antigen or analyte that also binds with the known antibody. Thisallows for the known antigen or analyte and another antigen or analyteto both compete for the binding site on the antibody. For example, alevetiracetam derivative that binds with an anti-levetiracetam antibodycan be combined with a sample suspected of containing levetiracetam, andthe derivative and levetiracetam compete for binding with theanti-levetiracetam antibody. The competition for binding with theantibody can then be used to determine whether or not levetiracetam ispresent in the sample, and can further be used to quantify the amount oflevetiracetam in the sample.

Linking Group

The term “linker” or “linking group” refers to a portion of a chemicalstructure that connects two or more substructures.

Conjugate

A conjugate is a molecule comprised of two or more substructures boundtogether, optionally through a linking group, to form a singlestructure. The binding can be made either by a direct connection (e.g. achemical bond) between the subunits or by use of a linking group. Forexample, a conjugate is a G6PDH enzyme or a label protein includingalkaline phosphatase, β-galactosidase and horse radish peroxidase or achemical label such as a fluorescent, luminescent or colorimetricmolecule or microparticle attached to a hapten or analyte analog orderivative.

Conjugation

Conjugation is any process wherein two subunits are linked together toform a conjugate. The conjugation process can be comprised of any numberof steps.

Derivative

As used herein, a “derivative” is a compound derived or obtained fromanother and containing essential elements of the parent substance. Thus,in one embodiment, the term derivative refers to a chemical compound ormolecule made from levetiracetam by one or more chemical reactions. Assuch, a derivative can be a compound with a structure similar to that oflevetiracetam or based on levetiracetam.

Hapten

Haptens are capable of binding specifically to corresponding antibodies,but usually do not themselves act as immunogens for preparation of theantibodies. Antibodies which recognize a hapten can be prepared againstcompounds comprised of the hapten linked to an immunogenic carrier.

Antibody

The term “anti-levetiracetam antibody” refers to antibodies that arecapable of specifically binding a levetiracetam epitope oflevetiracetam, a levetiracetam derivative, or a levetiracetam conjugate.“Anti-levetiracetam antibodies” include both polyclonal and monoclonalantibodies, as well as antigen-binding fragments thereof as definedabove. A “levetiracetam epitope” refers to an epitope that is present inlevetiracetam and in a levetiracetam derivative (e.g., a levetiracetamconjugate).

The term “binds specifically” or “specifically binds” in the context ofantibody binding, refers to high avidity and/or high affinity binding ofan antibody to a specific antigen, e.g., to levetiracetam. In specificbinding under appropriate conditions, antibody binding to levetiracetamis stronger than binding of the same antibody to any other epitope,particularly those which may be present in molecules in associationwith, or in the same sample, as the levetiracetam to be detected, e.g.,binds more strongly (e.g., higher affinity, higher avidity, or both) tolevetiracetam than to a non-levetiracetam epitope so that by adjustingbinding conditions the antibody binds almost exclusively tolevetiracetam or a levetiracetam moiety as present in a compound of thepresent disclosure (see Formula 1-3), and not to non-levetiracetammoieties that may be present in the sample. Antibodies which bindspecifically to levetiracetam may be capable of binding other antigensat a weak, yet detectable, level (e.g., 10% or less of the binding shownto levetiracetam). Such weak binding, or background binding, is readilydiscernible from the specific antibody binding to levetiracetam, e.g.,by use of appropriate controls. “Antibody activity” or “antibody bindingactivity” in the context of analyte binding assays generally refers tothe ability of an antibody to bind a specific antigen or analyte inpreference to other potential antigens or analytes via the antigencombining site located within a variable region of an immunoglobulin.

The term “antibody” includes a protein molecule having one or morepolypeptides substantially encoded by all or part of the recognizedimmunoglobulin genes. The recognized immunoglobulin genes, for examplein humans, include the kappa (κ), lambda (κ), and heavy chain geneticloci, which together comprise the myriad variable region genes, and theconstant region genes mu (μ), delta (δ), gamma (γ), epsilon (ε), andalpha (α) which encode the IgM, IgD, IgG, IgE, and IgA isotypesrespectively. Antibody herein is meant to include full length antibodiesand antibody fragments, and may refer to a natural antibody from anyorganism, an engineered antibody, or an antibody generated recombinantlyfor experimental, therapeutic, or other purposes as further definedbelow. Thus, the term “antibody raised against a compound” includes asynthesized antibody or compound having the same structure as anantibody raised against the compound The term “antibody” includesantibody fragments, as are known in the art, such as Fab, Fab′, F(ab′)₂,Fv, scFv, or other antigen-binding subsequences of antibodies, eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA technologies. The term “antibody” refers toboth monoclonal and polyclonal antibodies. Antibodies can beantagonists, agonists, neutralizing, inhibitory, or stimulatory.

As used herein, the term “polyclonal antibody” refers to a heterogeneousmixture of antibodies with a wide range of specificities and affinitiesto a given antigen or epitope. Thus, the polyclonal antibody, which canalso be referred to as polyclonal antibodies, can include a plurality ofantibodies, each distinguishable from the others, that bind or otherwiseinteract with an antigen. The term “polyclonal” refers to antibodiesoriginating from multiple progenitor cells. The different antibodiesthat comprise a polyclonal antibody can be produced or generated byinjecting an immunogen having an epitope into an animal and, after anappropriate time, collecting and optionally purifying the blood fractioncontaining the antibodies of interest. In producing antibodies, severalparameters can be considered with respect to the final use for thepolyclonal antibody. These parameters include the following: (1) thespecificity of the antibody (i.e., the ability to distinguish betweenantigens); (2) the avidity of the antibody (i.e., the strength ofbinding an epitope); and (3) the titer of the antibody, which determinesthe optimal dilution of the antibody in the assay system.

As used herein, the term “monoclonal antibody” refers to an antibodythat is isolated from a culture of normal antibody-producing cells andone unique progenitor cell. A monoclonal antibody can have a homogeneousbinding constant. The monoclonal antibodies include an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen.

The monoclonal antibodies include hybrid and recombinant antibodiesproduced by splicing a variable (including hypervariable) domain of ananti-inhibitor antibody with a constant domain, or a light chain with aheavy chain, or a chain from one species with a chain from anotherspecies, or fusions with heterologous proteins, regardless of species oforigin or immunoglobulin class or subclass designation, as well asantibody fragments, e.g., Fab, F(ab)2, and Fv 1, so long as they exhibitthe desired biological activity. For example, the monoclonal antibodiesmay be made by the hybridoma method first described by Kohler &Milstein, Nature, 256:495 (1975), or may be made by recombinant DNAmethods (U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may alsobe isolated from phage libraries generated using the techniquesdescribed in, e.g., McCafferty et al., Nature, 348:552-554 (1990).

Antigen

“Antigen”, as used herein, refers to a compound that binds specificallyto the variable region or binding site of an antibody. The term“antigen” and “immunogen” may in some cases be used interchangeably.

Epitope

The term “epitope” refers to a region of an antigen that interacts withan antibody molecule. An antigenic molecule can have one or moreepitopes that can be recognized by the same or different antibodies. Anepitope or epitopic moiety may comprise a unique chemical configurationof an antigen, hapten or a reactive ligand. The chemical configurationmay be a linear sequence of chemical composition or even a spatial arrayof chemical groups in the chemical configuration. An epitope is thechemical configuration that associates directly with the binding site inthe antibody molecule. The antibody and the chemical group, hapten orreacting ligand containing the epitope form the “specific binding pair.”

Immunogen

As used herein, the terms “immunogen” and “immunogenic” are meant torefer to substances capable of producing or generating an immuneresponse (e.g., antibody response) in an organism. An immunogen can alsobe antigen. In one embodiment, the immunogen has a fairly high molecularweight (e.g. greater than 10,000). Thus, a variety of macromoleculessuch as proteins, lipoproteins, polysaccharides, nucleic acids andteichoic acids can be coupled to a hapten in order to form an immunogenin accordance with the embodiments.

As used herein, the term “immunogenicity” refers to the ability of amolecule to induce an immune response, which is determined both by theintrinsic chemical structure of the injected molecule and by whether ornot the host animal can recognize the compound. Small changes in thestructure of an antigen can greatly alter the immunogenicity of acompound and have been used extensively as a general procedure toincrease the chances of raising an antibody, particularly againstwell-conserved antigens. For example, these modification techniqueseither alter regions of the immunogen to provide better sites for T-Cellbinding or expose new epitopes for B-cell binding.

Immunogenic Carrier

“Immunogenic Carrier”, “Carrier,” or “Immunogenic Moiety,” as UsedHerein, Refers to any material that when combined with a haptenstimulates an in vitro or in vivo immune response. A hapten becomes animmunogenic moiety when coupled to a carrier and as part of theimmunogen can induce an immune response and elicit the production ofantibodies that can bind specifically with the hapten. Immunogeniccarrier moieties include proteins, peptides (including polypeptides),glycoproteins, saccharides including complex polysaccharides, particles,nucleic acids, polynucleotides, and the like that are recognized asforeign and thereby elicit an immunologic response from the host.

Inhibitory Antibody

An antibody capable of inhibiting the activity of an enzyme or anenzyme-hapten conjugate upon binding an epitope present on the enzyme.

Accuracy

The term “accuracy” refers to the closeness of the agreement between theresult of a measurand and a true value of the measurand. The measurandis the substance measured or analyzed, the analyte or the ligandentering the binding reaction with the receptor or antibody.

Specificity

The term “specificity” or “selectivity” refers to the preferentialbinding of a ligand to a receptor (e.g., antibody). Thus, specificitymay refer, in one embodiment, to the degree that levetiracetam is boundselectively by an antibody. One measure of the specificity of a receptorto a ligand is crossreactivity. Compounds that cross-react are referredto as “crossreactants.” Crossreactants may occur as the result of thebiotransformation of levetiracetam by the human body to a metabolite,such as levetiracetam being biotransformed into 2-pyrrolidone-N-butyricacid. Anti-levetiracetam antibodies of the present disclosure includethose that bind an epitope of levetiracetam, but that do not detectablybind a metabolite of levetiracetam, such as a 2-pyrrolidone-N-butyricacid metabolite of levetiracetam.

Levetiracetam Derivative

A “levetiracetam derivative” as used in this disclosure refers to acompound sharing a core structure with levetiracetam and that cancompete with levetiracetam for binding to an anti-levetiracetam bindingpartner, such as an anti-levetiracetam antibody.

Certain compounds disclosed herein in connection with embodiments canexist in unsolvated forms as well as solvated forms, including hydratedforms. In general, the solvated forms are equivalent to unsolvated formsand are encompassed within the reference to the compounds set out in thepresent disclosure. Certain compounds disclosed herein in connectionwith embodiments may exist in multiple crystalline or amorphous forms.

Isolated

As used herein, the term “isolated,” when used in the context of anisolated compound, antibody, conjugate, etc., refers to a compound ofinterest (e.g., a compound as described herein, a conjugate as describedherein, an antibody as described herein, etc.) that is in an environmentdifferent from that in which the compound naturally occurs. “Isolated”is meant to include compounds of interest (e.g., a compound as describedherein, a conjugate as described herein, or an antibody as describedherein) that are within samples that are substantially enriched for thecompound of interest and/or in which the compound of interest ispartially or substantially purified. As used herein, the term“substantially pure” refers to a compound of interest that is removedfrom its natural environment and is at least 60% free, at least about75% free, at least about 90% free, at least about 95% free, at leastabout 98% free, or more than 98% free, from other components with whichit is naturally associated, and/or with it may be associated duringsynthesis or production.

Certain compounds disclosed herein in connection with embodimentspossess asymmetric carbon atoms (optical centers) or double bonds; theracemates, diastereomers, geometric isomers and individual isomers areencompassed within the scope of the embodiments.

The compounds may be prepared as a single isomer (e.g., enantiomer,cis-trans, positional, diastereomer) or as a mixture of isomers. In oneembodiment, the compounds are prepared as substantially a single isomer.Methods of preparing substantially isomerically pure compounds are knownin the art. For example, enantiomerically enriched mixtures and pureenantiomeric compounds can be prepared by using synthetic intermediatesthat are enantiomerically pure in combination with reactions that eitherleave the stereochemistry at a chiral center unchanged or result in itscomplete inversion. Alternatively, the final product or intermediatesalong the synthetic route can be resolved into a single stereoisomer.Techniques for inverting or leaving unchanged a particular stereocenter,and those for resolving mixtures of stereoisomers are well known in theart and it is well within the ability of one of skill in the art tochoose and appropriate method for a particular situation. See,generally, Furniss et al. (eds.), Vogel's Encyclopedia of PracticalOrganic Chemistry, 5th ed., Longman Scientific and Technical Ltd.,Essex, 1991, pp. 809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—. Use of a single dash (“-”) or double dash (“—” or “--”) refersto a single covalent bond, while use of “═” refers to a double bond. Thesymbol, )₂ or ₂(, when displayed with —S, indicates that the compoundinside the parenthesis may be present as a dimer forming a disulfidebond. The dimer may be reduced to a monomer.

Acyl or Alkanoyl

The term “acyl” or “alkanoyl” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof, havingthe stated number of carbon atoms and an acyl radical on at least oneterminus of the alkane radical. The “acyl radical” is the group derivedfrom a carboxylic acid by removing the —OH moiety therefrom.

Alkyl

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include divalent(“alkylene”) and multivalent radicals, having the number of carbon atomsdesignated (i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.”, where “heteroalkyl” refers to carbon chains having oneor more substitutions at one or more carbon atoms of the hydrocarbonchain fragment. Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl”. Certain alkyl groups include those containingbetween about one and about twenty five carbon atoms (e.g. methyl, ethyland the like).

Lower Alkyl

The term “lower alkyl” generally refers to a straight, branched, orcyclic hydrocarbon chain containing 8 or fewer carbon atoms, and cancontain from 1 to 8, from 1 to 6, or from 1 to 4 carbon atoms. Certain“lower alkyl” groups include methyl, ethyl, n-propyl, isopropyl,n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl and thelike. “Lower alkyls” can be optionally substituted at one or more carbonatoms of the hydrocarbon chain.

Alkoxy, Alkylamino and Alkylthio

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused to refer to those alkyl groups attached to the remainder of themolecule via an oxygen atom, an amino group, or a sulfur atom,respectively.

Heteroatom

By “heteroatom” is meant atoms other than a carbon which may be presentin a carbon backbone or a linear, branched or cyclic compound. Certainheteroatoms include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P)and silicon (Si). Heteroatoms can be present in their reduced forms,e.g., —OH, —NH, and —SH.

Heteroalkyl

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a straight or branched chain, or cycliccarbon-containing radical, or combinations thereof, having the statednumber of carbon atoms and at least one heteroatom which can be a memberselected from O, N, Si, P and S, wherein the nitrogen, phosphorous andsulfur atoms are optionally oxidized, and the nitrogen heteroatom isoptionally be quaternized. Normally heteroalkyl groups contain no morethan two heteroatoms linked in sequence. The heteroatom(s) O, N, P, Sand Si may be placed at any interior position of the heteroalkyl groupor at the position at which the alkyl group is attached to the remainderof the molecule. Examples include, but are not limited to,—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Generally, up to two heteroatomsmay be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃.

Heteroalkylene

Similarly, the term “heteroalkylene” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂—.

Cycloalkyl and Heterocycloalkyl

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

Aryl

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic moiety that can be a single ring or multiple rings (usuallyfrom 1 to 3 rings), which are fused together or linked covalently. Theterm “heteroaryl” refers to aryl groups (or rings) that contain from oneto four heteroatoms which are members selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl,2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedbelow.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Certain substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ where eachcan be independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of theembodiments includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 5-, 6-, or 7-membered ring. For example, —NR′R″is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ can beindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the embodiments includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. In theschemes that follow, the symbol X represents “R” as described above.

Amino and Amine Group

The term “amino” or “amine group” refers to the group —NR′R″ (orN⁺RR′R″) where R, R′ and R″ are independently selected from hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl,substituted aryl alkyl, heteroaryl, and substituted heteroaryl. Asubstituted amine is an amine group wherein R′ or R″ is other thanhydrogen. In a primary amino group, both R′ and R″ are hydrogen, whereasin a secondary amino group, either, but not both, R′ or R″ is hydrogen.In addition, the terms “amine” and “amino” can include protonated andquaternized versions of nitrogen, comprising the group —N⁺RR′R″ and itsbiologically compatible anionic counterions.

Polypeptide

“Polypeptide” as used herein is meant to encompass a polyaminoacid ofany length, and encompasses proteins, protein fragments and peptides.Polypeptides may be genetically encoded or synthetically produced.Polypeptides may also be modified, e.g., by post-translational and/orchemical modification(s).

Detectable Label

As used herein, a “detectable label” generally refers to an identifyingtag that can provide for a detectable signal, e.g., luminescence (e.g.,photoluminescence (e.g., fluorescence, phosphorescence),chemoluminescence (e.g., bioluminescence), microparticle aggregation orformation, radioactivity, immunodetection, enzymatic activity, and thelike.

Detectably Labeled Antibody

By “detectably labeled antibody” an antibody (which, as defined above,includes antigen-binding fragments, etc.) having an attached detectablelabel. The detectable label may be attached by chemical conjugation, butwhere the label is a polypeptide, it could alternatively be attached bygenetic engineering techniques. Methods for production of detectablylabeled proteins are well known in the art. Detectable labels may beselected from a variety of such labels known in the art, but normallyare radioisotopes, fluorophores, enzymes (e.g., horseradish peroxidase),or other moieties or compounds which either emit a detectable signal(e.g., radioactivity, fluorescence, color) or emit a detectable signalafter exposure of the label to its substrate. Various detectablelabel/substrate pairs (e.g., horseradish peroxidase/diaminobenzidine,avidin/streptavidin, luciferase/luciferin), methods for labelingantibodies, and methods for using labeled antibodies to detect anantigen are well known in the art.

Antibody-Analyte Complex and Antibody-Antigen Complex

“Antibody-analyte complex”, “antibody-antigen complex” generally refersto a complex that results following specific binding of an antibody andits antigen or analyte, e.g., between an anti-levetiracetam antibody andlevetiracetam (or a levetiracetam derivative, e.g., levetiracetamconjugate).

Assessing

The term “assessing” includes any form of measurement, and includesdetermining the presence or absence if an element. The terms“assessing”, “determining” (e.g., as in “determining the presence orabsence of”), “measuring”, “evaluating”, and “assaying” are usedinterchangeably and include quantitative and qualitative determinations.Assessing may be relative or absolute. “Assessing the presence of”includes determining the amount of something present, and/or determiningwhether it is present or absent. As used herein, the terms“determining,” “measuring,” and “assessing,” and “assaying” are usedinterchangeably and include both quantitative and qualitativedeterminations.

DETAILED DESCRIPTION OF EMBODIMENTS

Before embodiments are described, it is to be understood that thisinvention is not limited to particular embodiments described, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting, since the scope of the presentinvention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the embodiments. The upper and lower limits of these smallerranges may independently be included or excluded in the range, and eachrange where either, neither or both limits are included in the smallerranges is also encompassed within the embodiments, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the embodiments.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the embodiments, some methods and materialsare now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. It is understood that the presentdisclosure supersedes any disclosure of an incorporated publication tothe extent there is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aconjugate” includes a plurality of such conjugates and reference to “thesample” includes reference to one or more samples and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Immuno Assay

Currently, there are three classes of immunoassay, which are describedas follows: (1) antibody capture assays; (2) antigen capture assays; and(3) two-antibody sandwich assays. Additionally, it is contemplated thatnew immunoassays will be developed and will be capable of employing thehapten derivatives and antibodies that form the specific binding pair ofthe embodiments. Immunoassay or immunodiagnostic test systems measure aligand or target analyte, the measurand (e.g., levetiracetam), by usingthe selective binding properties of an antibody and a signal generatingsystem comprising a signal generating moiety that is responsive orreactive to the presence of antibody due to the binding of the antibodywith hapten conjugated to the signal generating moiety.

Homogeneous enzyme immunoassays depend on the availability ofenzyme-hapten conjugates whose enzyme activity can be strongly modulatedupon binding of an antibody raised against an epitope present on thehapten. In one aspect, the embodiments provide enzyme-hapten conjugatesand antibodies for conducting assays that are useful in homogeneousimmunoassays. “Homogeneous immunoassay”, as used herein, refers to anassay method where the complex is typically in solution and notseparated from unreacted reaction components, but instead the presenceof the complex is detected by a property which at least one of thereactants acquires or loses as a result of being incorporated into thecomplex. Homogeneous assays known in the art include systems involvingfluorochrome and fluorochrome quenching pairs on different reagents(U.S. Pat. Nos. 3,996,345; 4,161,515; 4,256,834 and 4,264,968); enzymeand enzyme inhibitor pairs on different reagents (U.S. Pat. Nos.4,208,479 and 4,233,401); chromophore and chromophore modifier pairs ondifferent reagents (U.S. Pat. No. 4,208,479); and latex agglutinationassays (U.S. Pat. Nos. 3,088,875; 3,551,555; 4,205,954 and 4,351,824).

The anti-levetiracetam antibodies, either monoclonal or polyclonal, canbe used in immunoassays for identifying the presence of levetiracetam ina biological sample, such as blood, plasma, serum, urine, tissue, andthe like. This can be beneficial for identifying or determiningpharmacokinetic and/or pharmacodynamic parameters for levetiracetam in apatient or patient population. Thus, the anti-levetiracetam antibodiescan be used in immunodiagnostic assays so that the assays can beconfigured for identifying the presence and optionally quantifying theamount of levetiracetam. Additionally, the immunodiagnostic assays canuse levetiracetam derivatives in accordance with embodiments.

Levetiracetam Derivatives

A certain embodiment is a compound of Formula 1 shown below:

wherein R₁ is —Y—Z, and Y is a linking group and Z is selected from thegroup consisting of hydrogen, —OH, —SH, —S-acyl; —O-lower alkyl,halogen, —NH₂, -epoxy, -maleimidyl, haloacetamide, carboxyl, activatedcarboxyl, an immunogenic carrier, a protein, and a label, and includingacid salts thereof. Activated carboxyl includes all activated forms ofcarboxyl including, but not limited to, hydroxysuccinimidyl,succinimidyl, carbonate, and anhydride.

In certain embodiments, in Formula 1, Z is selected from the groupconsisting of hydrogen, —OH, —SH, —S-acyl; —O-lower alkyl, halogen, NH₂,-epoxy, -maleimidyl, haloacetamide, carboxyl, and activated carboxyl. Incertain embodiments, Z is selected from the group consisting of —SH,halogen, NH₂, -maleimidyl, carboxyl, and activated carboxyl. In certainembodiments, Z is halogen. In certain embodiments, Z is bromo. Incertain embodiments, Z is carboxyl. In certain embodiments, Z is —NH₂.In certain embodiments, Z is —SH.

In certain embodiments, in Formula 1, Z is an immunogenic carrier.Examples of immunogenetic carriers include proteins, peptides,glycoproteins, saccharides, particles, nucleic acids, andpolynucleotides. In certain embodiments, Z is an immunogenic carrierselected from hemocyanins, globulins, albumins, and polysaccharides.Examples of certain immunogenic carrier include keyhole limpethemocyanin (KLH), bovine serum albumin (BSA), or human serum albumin(HSA). In certain embodiments, Z is an immunogenic carrier selected fromBSA and KLH.

In certain embodiments, in Formula 1, Z is a protein. Examples ofcertain proteins that are also immunogenic carriers include keyholelimpet hemocyanin (KLH), bovine serum albumin (BSA), or human serumalbumin (HSA).

In certain embodiments, in Formula 1, Z is a label. Examples of labelsfor Z include, but are not limited to, isotopic labels and non-isotopicsignal generating moieties. Examples of non-isotopic signal generatingmoieties include fluorophores and enzymes, which are described infurther detail below.

Certain fluorophores include, but are not limited to, naphthalenederivatives (e.g. dansyl chloride), anthracene derivatives (e.g.N-hydroxysuccinimide ester of anthracene propionate), pyrene derivatives(e.g. N-hydroxysuccinimide ester of pyrene butyrate), fluoresceinderivatives (e.g. fluorescein isothiocyanate), rhodamine derivatives(e.g. rhodamine isothiocyanate), phycoerythin, and Texas Red.

Certain enzymes include, but are not limited to, alkaline phosphatase,horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase,lactate dehydrogenase, β-galactosidase, and urease. Also, a geneticallyengineered fragment of an enzyme may be used, such as the donor andacceptor fragment of β-galactosidase utilized in CEDIA immunoassays. Ina certain embodiment, the enzyme is glucose-6-phosphate dehydrogenase(G6PDH), alkaline phosphatase, β-galactosidase, and horseradishperoxidase. In a certain embodiment, the enzyme is glucose-6-phosphatedehydrogenase (G6PDH).

In certain embodiments, in Formula 1, Z is a halogen, such as bromo; andY is a linker comprising 3 carbon atoms and one nitrogen atom. Incertain embodiments, Formula 1 is a compound of the formula:

In certain embodiments, in Formula 1, Z is a halogen, such as bromo; andY is a linker comprising 4-5 carbon atoms and one nitrogen atom. Incertain embodiments, Formula 1 is a compound of the formula:

In certain embodiments, in Formula 1, Z is selected from the groupconsisting of —SH, halogen, NH₂, -maleimidyl, carboxyl, and activatedcarboxyl; and Y is a linker comprising 1-5 carbon atoms and zero or onenitrogen atom. In certain embodiments, in Formula 1, Z is selected fromthe group consisting of —SH, halogen, NH₂, -maleimidyl, carboxyl, andactivated carboxyl; and Y is —(CH₂)n-C(O)NH—(CH₂)n-, where each n is aninteger from one to ten. In certain embodiments, in Formula 1, Z isselected from the group consisting of —SH, halogen, NH₂, -maleimidyl,carboxyl, and activated carboxyl; and Y is —(CH₂)n-C(O)NH—(CH₂)n-, whereeach n is one or two. In certain embodiments, in Formula 1, Z isselected from the group consisting of —SH, halogen, NH₂, -maleimidyl,carboxyl, and activated carboxyl; and Y is —(CH₂)n-, where n is aninteger from one to ten. In certain embodiments, in Formula 1, Z isselected from the group consisting of —SH, halogen, NH₂, -maleimidyl,carboxyl, and activated carboxyl; and Y is —(CH₂)—.

A certain embodiment is a compound of Formula 2 shown below:

wherein R₂ is —Y—Z, and Y is a linking group and Z is selected from thegroup consisting of hydrogen, —OH, —SH, —S-acyl; —O-lower alkyl,halogen, —NH₂, -epoxy, -maleimidyl, haloacetamide, carboxyl, activatedcarboxyl, an immunogenic carrier, a protein, and a label, and includingacid salts thereof. Activated carboxyl includes all activated forms ofcarboxyl including, but not limited to, hydroxysuccinimidyl,succinimidyl, carbonate, and anhydride.

In certain embodiments, in Formula 2, Z is selected from the groupconsisting of hydrogen, —OH, —SH, —S-acyl; —O-lower alkyl, halogen, NH₂,-epoxy, -maleimidyl, haloacetamide, carboxyl, and activated carboxyl. Incertain embodiments, Z is selected from the group consisting of —SH,halogen, NH₂, -maleimidyl, carboxyl, and activated carboxyl. In certainembodiments, Z is halogen. In certain embodiments, Z is bromo. Incertain embodiments, Z is carboxyl. In certain embodiments, Z is —NH₂.In certain embodiments, Z is —SH.

In certain embodiments, in Formula 2, Z is an immunogenic carrier.Examples of immunogenetic carriers include proteins, peptides,glycoproteins, saccharides, particles, nucleic acids, andpolynucleotides. In certain embodiments, Z is an immunogenic carrierselected from hemocyanins, globulins, albumins, and polysaccharides.Examples of certain immunogenic carrier include keyhole limpethemocyanin (KLH), bovine serum albumin (BSA), or human serum albumin(HSA). In certain embodiments, Z is an immunogenic carrier selected fromBSA and KLH.

In certain embodiments, in Formula 2, Z is a protein. Examples ofcertain proteins that are also immunogenic carriers include keyholelimpet hemocyanin (KLH), bovine serum albumin (BSA), or human serumalbumin (HSA).

In certain embodiments, in Formula 2, Z is a label. Examples of labelsfor Z include, but are not limited to, isotopic labels and non-isotopicsignal generating moieties. Examples of non-isotopic signal generatingmoieties include fluorophores and enzymes, which are described infurther detail below.

Certain fluorophores include, but are not limited to, naphthalenederivatives (e.g. dansyl chloride), anthracene derivatives (e.g.N-hydroxysuccinimide ester of anthracene propionate), pyrene derivatives(e.g. N-hydroxysuccinimide ester of pyrene butyrate), fluoresceinderivatives (e.g. fluorescein isothiocyanate), rhodamine derivatives(e.g. rhodamine isothiocyanate), phycoerythin, and Texas Red.

Certain enzymes include, but are not limited to, alkaline phosphatase,horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase,lactate dehydrogenase, β-galactosidase, and urease. Also, a geneticallyengineered fragment of an enzyme may be used, such as the donor andacceptor fragment of β-galactosidase utilized in CEDIA immunoassays. Ina certain embodiment, the enzyme is glucose-6-phosphate dehydrogenase(G6PDH), alkaline phosphatase, β-galactosidase, and horseradishperoxidase. In a certain embodiment, the enzyme is glucose-6-phosphatedehydrogenase (G6PDH).

In certain embodiments, in Formula 2, Z is selected from the groupconsisting of —SH, halogen, NH₂, -maleimidyl, carboxyl, and activatedcarboxyl; and Y is a linker comprising 1-5 carbon atoms and zero or onenitrogen atom. In certain embodiments, in Formula 1, Z is selected fromthe group consisting of —SH, halogen, NH₂, -maleimidyl, carboxyl, andactivated carboxyl; and Y is —(CH₂)n-C(O)NH—(CH₂)n-, where each n is aninteger from one to ten. In certain embodiments, in Formula 1, Z isselected from the group consisting of —SH, halogen, NH₂, -maleimidyl,carboxyl, and activated carboxyl; and Y is —(CH₂)n-C(O)NH—(CH₂)n-, whereeach n is one or two. In certain embodiments, in Formula 1, Z isselected from the group consisting of —SH, halogen, NH₂, -maleimidyl,carboxyl, and activated carboxyl; and Y is —(CH₂)n-, where n is aninteger from one to ten. In certain embodiments, in Formula 1, Z isselected from the group consisting of —SH, halogen, NH₂, -maleimidyl,carboxyl, and activated carboxyl; and Y is —(CH₂)—.

A certain embodiment is a compound of Formula 3 shown below:

wherein R₃ is —Y—Z, and Y is a linking group and Z is selected from thegroup consisting of hydrogen, —OH, —SH, —S-acyl; —O-lower alkyl,halogen, —NH₂, -epoxy, -maleimidyl, haloacetamide, carboxyl, activatedcarboxyl, an immunogenic carrier, a protein, and a label, and includingacid salts thereof. Activated carboxyl includes all activated forms ofcarboxyl including, but not limited to, hydroxysuccinimidyl,succinimidyl, carbonate, and anhydride.

In certain embodiments, in Formula 3, Z is selected from the groupconsisting of hydrogen, —OH, —SH, —S-acyl; —O-lower alkyl, halogen, NH₂,-epoxy, -maleimidyl, haloacetamide, carboxyl, and activated carboxyl. Incertain embodiments, Z is selected from the group consisting of —SH,halogen, NH₂, -maleimidyl, carboxyl, and activated carboxyl. In certainembodiments, Z is halogen. In certain embodiments, Z is bromo. Incertain embodiments, Z is carboxyl. In certain embodiments, Z is —NH₂.In certain embodiments, Z is —SH.

In certain embodiments, in Formula 3, Z is an immunogenic carrier.Examples of immunogenetic carriers include proteins, peptides,glycoproteins, saccharides, particles, nucleic acids, andpolynucleotides. In certain embodiments, Z is an immunogenic carrierselected from hemocyanins, globulins, albumins, and polysaccharides.Examples of certain immunogenic carrier include keyhole limpethemocyanin (KLH), bovine serum albumin (BSA), or human serum albumin(HSA). In certain embodiments, Z is an immunogenic carrier selected fromBSA and KLH.

In certain embodiments, in Formula 3, Z is a protein. Examples ofcertain proteins that are also immunogenic carriers include keyholelimpet hemocyanin (KLH), bovine serum albumin (BSA), or human serumalbumin (HSA).

In certain embodiments, in Formula 3, Z is a label. Examples of labelsfor Z include, but are not limited to, isotopic labels and non-isotopicsignal generating moieties. Examples of non-isotopic signal generatingmoieties include fluorophores and enzymes, which are described infurther detail below.

Certain fluorophores include, but are not limited to, naphthalenederivatives (e.g. dansyl chloride), anthracene derivatives (e.g.N-hydroxysuccinimide ester of anthracene propionate), pyrene derivatives(e.g. N-hydroxysuccinimide ester of pyrene butyrate), fluoresceinderivatives (e.g. fluorescein isothiocyanate), rhodamine derivatives(e.g. rhodamine isothiocyanate), phycoerythin, and Texas Red.

Certain enzymes include, but are not limited to, alkaline phosphatase,horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase,lactate dehydrogenase, β-galactosidase, and urease. Also, a geneticallyengineered fragment of an enzyme may be used, such as the donor andacceptor fragment of β-galactosidase utilized in CEDIA immunoassays. Ina certain embodiment, the enzyme is glucose-6-phosphate dehydrogenase(G6PDH), alkaline phosphatase, β-galactosidase, and horseradishperoxidase. In a certain embodiment, the enzyme is glucose-6-phosphatedehydrogenase (G6PDH).

In certain embodiments, in Formula 3, Z is selected from the groupconsisting of —SH, halogen, NH₂, -maleimidyl, carboxyl, and activatedcarboxyl; and Y is a linker comprising 1-5 carbon atoms and zero or onenitrogen atom. In certain embodiments, in Formula 1, Z is selected fromthe group consisting of —SH, halogen, NH₂, -maleimidyl, carboxyl, andactivated carboxyl; and Y is —(CH₂)n-C(O)NH—(CH₂)n-, where each n is aninteger from one to ten. In certain embodiments, in Formula 1, Z isselected from the group consisting of —SH, halogen, NH₂, -maleimidyl,carboxyl, and activated carboxyl; and Y is —(CH₂)n-C(O)NH—(CH₂)n-,wherein each n is one or two. In certain embodiments, in Formula 1, Z isselected from the group consisting of —SH, halogen, NH₂, -maleimidyl,carboxyl, and activated carboxyl; and Y is —(CH₂)n-, where n is aninteger from one to ten. In certain embodiments, in Formula 1, Z isselected from the group consisting of —SH, halogen, NH₂, -maleimidyl,carboxyl, and activated carboxyl; and Y is —(CH₂)—.

Linking Group

A linking group or linker has at least 1 uninterrupted chain of atomsextending between the substructures, as depicted as Y in Formulae 1-3.The atoms of a linking group are themselves connected by chemical bonds.The number of atoms in a linking group is determined by counting theatoms other than hydrogen. In some embodiments, the linker is a part ofthe compound of the embodiments. In some embodiments, the linker canprovide a connection between, for example, the levetiracetam derivativeof Formula 1 and Z; the levetiracetam derivative of Formula 2 and Z; andthe levetiracetam derivative of Formula 3 and Z.

The compounds may be connected to other species by bonding between areactive functional group on the compound or a linker attached to thecompound, and a reactive functional group of complementary reactivity onthe other species. A linker may be, for example, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl and combinations thereof. A linker may alsoinclude cyclic and/or aromatic groups as part of the chain or as asubstitution on one of the atoms in the chain. In one embodiment, thelinker may be used to provide an available site on a hapten forconjugating the hapten with, for example, a label, carrier, immunogeniccarrier or the like. The linker molecule may be used to connect(conjugate or couple) the ligand, hapten, epitope or epitopic moiety toits immunogenic carrier or signal generating moiety and to display theligand, hapten, epitope or epitopic moiety for binding to the receptoror antibody. The length of the linker may be varied by those skilled inthe art to accomplish the desired outcome in producing the immunogen orthe signal generating system.

In certain embodiments, the linking group comprises 1-15 carbon atomsand/or 0-6 heteroatoms. In certain embodiments, the linking group isselected from the group consisting of —(CH₂)n-C(O)—, or —C(O)(CH₂)n- or—C(O)(CH₂)n-NHC(O)—, or —C(O)(CH₂)n-NHC(O)(CH₂)n-, or —(CH₂)nSCH₂C(O)—,or —(CH₂)n-C(O)NH—(CH₂)n-, or —(CH₂)n-NH—C(O)—, or—(CH₂)n-NH—C(O)—(CH₂)n-, or —C(O)—(CH₂)n-, or —(CH₂)n-NH—; and n is aninteger from 1 to 10, and including acid salts thereof.

In certain embodiments, the linking group is —(CH₂)n-C(O)NH—(CH₂)n-,where each n is an integer from one to ten. In certain embodiments, thelinking group is —(CH₂)n-C(O)NH—(CH₂)n-, where each n is one or two. Incertain embodiments, the linking group is —(CH₂)n-, where n is aninteger from one to ten. In certain embodiments, the linking group is—(CH₂)—.

In certain embodiments, the linking group is—(CH₂)n-C(O)N(CH₂)n(CH₃)—(CH₂)n-, where each n is an integer from one toten. In certain embodiments, the linking group is—(CH₂)n-C(O)N(CH₂)n(CH₃)—(CH₂)n-, where each n is one or two.

In certain embodiments, the linking group is —(CH₂)n-C(O)N(CH₃)—(CH₂)n-,where each n is an integer from one to ten. In certain embodiments, thelinking group is —(CH₂)n-C(O)N(CH₃)—(CH₂)n, where each n is one or two.

In certain embodiments, the linking group comprises 10-15 carbon atomsand/or 0-6 heteroatoms.

Additionally, linkers that link a carrier to a hapten can comprisemodified or unmodified nucleotides, nucleosides, polymers, sugars andother carbohydrates, polyethers, such as for example, polyethyleneglycols, polyalcohols, polypropylenes, propylene glycols, mixtures ofethylene and propylene glycols, polyalkylamines, polyamines such asspermidine, polyesters such as poly(ethyl acrylate),polyphosphodiesters, and alkylenes. An example of an operative group andits linker is cholesterol-TEG-phosphoramidite, wherein the cholesterolis the operative group and the tetraethylene glycol and phosphate serveas linkers.

In one embodiment, the immunogenic carrier is a protein. Proteincarriers can be highly soluble and include functional groups that couldfacilitate easy conjugation with a hapten molecule. In a certainembodiment, the immunogenic carrier is a member selected from keyholelimpet hemocyanin (KLH), bovine serum albumin (BSA) and human serumalbumin (HSA). Keyhole limpet hemocyanin is an oxygen-carrying proteinof the marine keyhole limpet, is extremely large and exhibits increasedimmunogenicity when it is disassociated into subunits. BSA is a highlysoluble protein containing numerous functional groups suitable forconjugation.

Derivatives

Derivatives of levetiracetam in accordance with some embodiments can beused to compete for binding with a receptor including an antibody thatrecognizes both the derivative and levetiracetam. Also, a derivative caninclude an operative group coupled to levetiracetam through a linker.Thus, the embodiments provide for levetiracetam derivatives linked to,for example, an immunogenic carrier and/or a signal generating moiety asoperative groups.

Signal Producing System

The signal producing system is utilized in assays for analytes and mayhave one or more components, at least one component being a mutantG6PDH. The signal producing system generates a signal that relates tothe presence or amount of analyte in a sample. The signal producingsystem includes all of the reagents required to produce a measurablesignal. In one aspect, the G6PDH or a label protein including alkalinephosphatase, B-galactosidase and horse radish peroxidase is conjugatedto a sbp member analogous to the analyte.

Other components of the signal producing system can include substrates,enhancers, activators, chemiluminescent compounds, cofactors,inhibitors, scavengers, metal ions, specific binding substances requiredfor binding of signal generating substances, coenzymes, substances thatreact with enzymic products, other enzymes and catalysts, and the like.

The signal producing system provides a signal detectable by externalmeans, normally by measurement of electromagnetic radiation, desirablyby visual examination. For the most part, the signal producing systemmay include a chromophoric substrate and mutant G6PDH enzyme, wherechromophoric substrates are enzymatically converted to dyes which absorblight in the ultraviolet or visible region.

Substantial Change in Enzyme Activity

A change in activity of an enzyme sufficient to allow detection of ananalyte when the enzyme is used as a label in an assay for the analyte.Typically, the enzyme's activity is reduced 10-100% preferably 20-99%,more preferably 30-95%.

Ancillary Materials

Various ancillary materials will frequently be employed in an assay inaccordance with the embodiments. For example, buffers will normally bepresent in the assay medium, as well as stabilizers for the assay mediumand the assay components. Frequently, in addition to these additives,additional proteins may be included, such as albumins, or surfactants,particularly non-ionic surfactants, binding enhancers, e.g.,polyalkylene glycols, or the like.

Anti-Levetiracetam Antibodies

As noted above, the term “antibody” as used in the context of thepresent disclosure, refers to a specific binding partner of an analyte(e.g., levetiracetam), and is meant to encompass whole antibodies aswell as antigen-binding fragments thereof (such as, for example,F(ab′)2, Fab′, Fab and Fv), naturally occurring antibodies, hybridantibodies, chimeric antibodies, single-chain antibodies, and antibodyfragments that retain antigen binding specificity, and the like.Antibodies can be of any class (e.g., IgM, IgG, IgA, IgE; frequentlyIgG) and generated from any source (although usually non-human, usuallya non-human mammal such as a rabbit, mouse, rat, goat, etc.). Thus,“antibody” is meant to encompass not only intact immunoglobulinmolecules, but also such fragments and derivatives of immunoglobulinmolecules as may be prepared by techniques known in the art, andretaining the antibody activity of an intact immunoglobulin.

Antibodies may be derived from polyclonal compositions monoclonalcompositions. As noted above, “antibodies” is also meant to encompasssingle chain antibodies or scFvs, where such recombinantly producedantibody fragments retain the binding characteristics of the aboveantibodies. Recombinantly produced antibody fragments within the meaningof “antibody” generally include at least the VH and VL domains of thesubject antibodies, so as to retain the binding characteristics of thesubject antibodies. These recombinantly produced antibody fragments maybe readily prepared using any convenient methodology, such as themethodology disclosed in U.S. Pat. Nos. 5,851,829 and 5,965,371; thedisclosures of which are herein incorporated by reference.

Anti-levetiracetam antibodies include those that bind one or morelevetiracetam epitopes. Anti-levetiracetam antibodies may bind one ormore of unconjugated levetiracetam, a levetiracetam derivative, alevetiracetam conjugate, or any combination thereof. The disclosureencompasses an antibody reactive to a common epitope present inlevetiracetam and compounds shown in formulas 1-3. Such antibodies canthus bind a levetiracetam epitope as present in levetiracetam and alevetiracetam moiety as present in a compound of any one of Formulae 1to 3.

Producing Anti-Levetiracetam Antibodies

Anti-levetiracetam antibodies can be prepared by using an immunogeniclevetiracetam conjugate described herein and applying methods forantibody production that are well known in the art. For examples ofgeneral techniques used in raising, purifying and modifying antibodies,and the design and execution of immunoassays, the reader is referred toHandbook of Experimental Immunology (D. M. Weir & C. C. Blackwell,eds.); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); David Wild, ed., The Immunoassay Handbook (Stockton Press N.Y.,1994); and R. Masseyeff, W. H. Albert, and N. A. Staines, eds., Methodsof Immunological Analysis (Weinheim: VCH Verlags gesellschaft mbH,1993).

Antibodies obtained using any of the disclosed techniques are screenedor purified not only for their ability to react with levetiracetam, butfor a low cross-reactivity with potential interfering substances.“Cross-reactivity” may be determined in a quantitative immunoassay byestablishing a standard curve using known dilutions of the targetanalyte, levetiracetam. The standard curve is then used to calculate theapparent concentration of the interfering substance present in variousknown amounts in samples assayed under similar condition. Thecross-reactivity can be calculated as the apparent concentration dividedby the actual concentration multiplied by 100. A certain immunoassay fordetermining cross-reactivity is a homogeneous enzyme immunoassay using awild type G6PDH as described in U.S. Pat. No. 3,817,837 or mutant G6PDHengineered to contain a cysteine per subunit as described in U.S. Pat.Nos. 6,033,890, 6,090,567 and 6,455,288. Furthermore, thecross-reactivity can be determined in the same type of immunoassay inwhich the antibody will ultimately be used.

Producing Polyclonal Antibodies

Polyclonal antibodies that bind levetiracetam may be raised byadministration of an immunogenic levetiracetam conjugate to an animalhost, usually mixed with an adjuvant. Any animal host which producesantibodies can be used. The immunogen is conveniently prepared forinjection by rehydrating lyophilized immunogen to form a solution orsuspension. Certain adjuvants are water-in-oil immersions, particularlyFreund's complete adjuvant for the first administration, and Freund'sincomplete adjuvant for booster doses. The preparation is typicallyadministered in a variety of sites, and typically in two or more dosesover a course of at least 4 weeks. Serum is harvested and tested for thepresence of anti-levetiracetam antibody using a levetiracetam-proteinconjugate or other levetiracetam conjugates in a standard immunoassay orprecipitation reaction.

Methods for purifying specific antibodies having a desired bindingspecificity from a polyclonal antiserum are known in the art. Aparticularly effective method is affinity purification using a column oflevetiracetam conjugated to a solid phase. One manner of preparing alevetiracetam column is to conjugate levetiracetam or a levetiracetamderivative to a protein other than the protein used in the immunogen,and then attach the conjugate to a commercially available activatedresin, such as CNBr-activated SEPHAROSE™. The anti-levetiracetamantibody is passed over the column, the column is washed, and theantibody is eluted with a mild denaturing buffer such as 0.1 M glycine,0.2 M NaCl, pH 2.5.

Producing Monoclonal Antibodies

Anti-levetiracetam monoclonal antibodies are prepared by a number ofdifferent techniques known in the art. For example, for hybridomatechnology, the reader is referred generally to Harrow E, Lane D., 1988,Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory Press, and Methods in Enzymology, 73B:3 (1981). Onecommon way to produce monoclonal antibodies is to immortalize and clonea splenocyte or other antibody-producing cell recovered from an animalthat has been immunized against levetiracetam as described earlier. Theclone is immortalized by a procedure such as fusion with a non-producingmyeloma, by transfecting with Epstein Barr Virus, or transforming withoncogenic DNA. The treated cells are cloned and cultured, and clones areselected that produce antibody of the desired specificity. Specificitytesting may be performed on culture supernatants by a number oftechniques, such as using the immunizing antigen as the detectingreagent in an immunoassay. A supply of monoclonal antibody from theselected clone can then be purified from a large volume of culturesupernatant, or from the ascites fluid of suitably prepared host animalsinjected with the clone. The antibody may be tested for activity as rawsupernatant or ascites, and is optionally purified using standardbiochemical preparation techniques such as ammonium sulfateprecipitation, ion exchange chromatography, and gel filtrationchromatography.

Producing Fragments and other Derivatives of Immunoglobulins

Fragments and other derivatives of immunoglobulins can be prepared bymethods of standard protein chemistry, for example, subjecting theantibody to cleavage with a proteolytic enzyme such as pepsin, papain,or trypsin; and reducing disulfide bonds with such reagents asdithiothreitol. Genetically, engineered variants of intactimmunoglobulin can be produced by obtaining a polynucleotide encodingthe antibody, and applying the general methods of molecular biology tosplice encoding sequences or introduce mutations and translate thevariant. Antibodies that are engineered variants of particular interestinclude chimeric and humanized antibodies, Fab-like fragments,single-chain variable region fragments (scFv), and diabodies.

Detectably Labeled Anti-Levetiracetam Antibodies

The anti-levetiracetam antibodies may also be labeled in order tofacilitate detection. A variety of protein labeling schemes are known inthe art and may be employed, the particular scheme and label chosenbeing the one most convenient for the intended use of the antibody, e.g.immunoassay.

Examples of labels include labels that permit both the direct andindirect measurement of the presence of the antibody. Examples of labelsthat permit direct measurement of the antibody include radiolabels, suchas ³H or ¹²⁵I, fluorescers, dyes, microparticles, beads,chemiluminescers, colloidal particles, and the like. Examples of labelswhich permit indirect measurement of the presence of the antibodyinclude enzymes where a substrate may provide for a colored orfluorescent product. For example, the antibodies may be labeled with acovalently bound enzyme capable of providing a detectable product signalafter addition of suitable substrate. Instead of covalently binding theenzyme to the antibody, the antibody may be modified to comprise a firstmember of specific binding pair which specifically binds with a secondmember of the specific binding pair that is conjugated to the enzyme,e.g. the antibody may be covalently bound to biotin and the enzymeconjugate to streptavidin. Examples of suitable enzymes for use inconjugates include horseradish peroxidase, alkaline phosphatase, malatedehydrogenase and the like. Where not commercially available, suchantibody-enzyme conjugates are readily produced by techniques known tothose skilled in the art.

Immunoassays

The present disclosure provides immunoassay methods for assessing thepresence or absence of levetiracetam in a sample of interest. Due tovarious factors, including the pronounced inter-individual variabilityin levetiracetam pharmacokinetics, immunoassays to assess levetiracetamstatus are of interest. Since levetiracetam is excreted renallyunchanged and is not metabolized or at least does not produce detectablemetabolites. Therefore, there are no pharmacogenomic issues that affectlevetiracetam concentration and it is not subject to significantpharmacokinetic drug interactions with other drugs.

Immunoassays of the present disclosure can be of a variety of formats.The immunoassays may be separation immunoassays (also known asheterogeneous immunoassays) or homogeneous immunoassays. Furthermore,the immunoassays may be qualitative or quantitative. Assays of thisdisclosure include both sandwich and competition assays. Theimmunoassays may embody assays that are neither sandwich nor competitionassays, as in certain assays involving immunoprecipitation.

In general, the immunoassays of the present disclosure for detecting thepresence or absence of levetiracetam in a sample can be conducted byadding, to a reaction mixture, (i) a sample suspected of containinglevetiracetam and (ii) an anti-levetiracetam antibody capable of forminga complex of levetiracetam that may be present in the sample and theantibody; and detecting the presence or absence of the complex. Thepresence or absence of said complex is indicative of the presence orabsence of levetiracetam in said sample. Moreover, the amount of complexformed can be assessed to determine the concentration of levetiracetampresent in the sample (e.g., to provide an assessment of serum or tissueconcentration of levetiracetam in a subject from whom the sample wasobtained). The presence and/or amount of complex can be assesseddirectly (e.g., by detecting bound antibody in the complex) orindirectly (e.g., by assessing activity of an enzyme in a levetiracetamenzyme conjugate, where when the levetiracetam enzyme conjugate is notbound to antibody, a detectable signal is generated, indicating that theanti-levetiracetam antibody in the reaction mixture has been bound bylevetiracetam from the sample).

In general, the immunoassays of the disclosure entail combining thesample with an anti-levetiracetam antibody under conditions that permitthe formation of a stable complex between the analyte to be tested andthe antibody.

Assays may be performed in solution or may use a solid (insoluble)support (e.g. polystyrene, nitrocellulose, or beads), using any standardmethods (e.g., as described in Current Protocols in Immunology, Coliganet al., ed.; John Wiley & Sons, New York, 1992). Typical methods includeELISAs (enzyme-linked immunosorbent assays), IRMAs (immunoradiometricassays), and RIAs (radioimmunoassays).

Where the assay is performed in solution, the test sample (and,optionally a control sample) is incubated with an anti-levetiracetamantibody for a time period sufficient to allow formation of analyte andaffinity reagent complexes, for example, between about 0.1 hrs up to 24hrs, or more. As previously noted, the anti-levetiracetam antibody mayinclude a detectable label (e.g. radionuclide, fluorescer, or enzyme).The sample is then treated to separate thelevetiracetam-anti-levetiracetam antibody complexes from excess,unreacted anti-levetiracetam antibody (e.g. by addition of ananti-anti-levetiracetam antibody (e.g., anti-immunoglobulin antiserum)followed by centrifugation to precipitate the complexes, or by bindingto an affinity surface such as a second, unlabelled anti-levetiracetamantibody fixed to a solid substrate such as Sepharose® or a plasticwell). Detection of anti-levetiracetam antibody bound to a levetiracetammay be achieved in a variety of ways well known in the art. Ifnecessary, a substrate for the detectable label may be added to thesample.

Where the assay uses a solid support, the support can have ananti-levetiracetam antibody (or levetiracetam conjugate) bound to asupport surface. Binding of the assay reagent facilitates the stable,wash-resistant binding of levetiracetam which may be present in thesample (or anti-levetiracetam antibody that is not bound tolevetiracetam from the sample, and is present in the reaction mixture,as in a competitive binding assay) to the solid support via specificbinding to the anti-levetiracetam antibody. The insoluble supports maybe any compositions to which antibodies or suitable levetiracetamconjugates can be bound, which is readily separated from solublematerial, and which is otherwise compatible with the overall method ofdetection of anti-levetiracetam antibody a sample.

The surface of such supports may be solid or porous and of anyconvenient shape. Examples of suitable insoluble supports to which theanti-levetiracetam antibody is bound include beads, e.g. magnetic beads,membranes and microtiter plates. These can be composed of glass, plastic(e.g. polystyrene), polysaccharides, nylon or nitrocellulose.

Assay reagents can include the anti-levetiracetam antibodies asdisclosed herein, as well as anti-anti-levetiracetam antibodies, whichmay be optionally detectably labeled. Methods for binding antibodies orother proteins to solid supports are well known in the art. Afterbinding of an assay reagent to the support, the support may be treatedwith a blocking agent, which binds to the support in areas not occupiedby the assay reagent Suitable blocking agents include non-interferingproteins such as bovine serum albumin, casein, gelatin, and the like.Alternatively, several detergents at non-interfering concentrations,such as Tween, NP40, TX100, and the like may be used. Such blockingtreatment reduces nonspecific binding.

Qualitative and Quantitative Methods

Assays of this disclosure include both qualitative and quantitativeassays. Typical quantitative methods involve mixing an analyte with apre-determined amount of the reagent antibody, and correlating theamount of complex formed with the amount of analyte in the originalsample using a relationship determined using standard samples containingknown amounts of analyte in the range expected for the sample to betested. In a qualitative assay, sufficient complex above or below athreshold level established by samples known to contain or be free ofanalyte establish the assay result. Unless otherwise stated, the act of“measuring” or “determining” in this disclosure refers alternately toqualitative and quantitative determination.

Samples

Samples may be biological samples taken from subjects suspected of beingadministered levetiracetam.

As used herein, a “biological sample” refers to a sample of tissue orfluid isolated from a subject, which in the context of the embodimentsgenerally refers to samples suspected of containing levetiracetam, whichsamples, after optional processing, can be analyzed in an in vitroassay. Certain samples of interest include, but are not necessarilylimited to, a “blood sample” (which as used herein is meant to includewhole blood, plasma, serum, and the like), fecal matter, urine, tears,sweat saliva, milk, organs, biopsies, secretions of the intestinal andrespiratory tracts, vitreous humor, and fluids obtainable during autopsy(such as cerebrospinal fluid). It should be noted that a “blood-derivedsample” refers to a sample that is prepared from blood or a fractionthereof, e.g., plasma or serum. Respiratory secretions (e.g., samplesobtained from fluids or tissue of nasal passages, lung, and the like),“Human serum”, as used herein, refers to the aqueous portion of humanblood remaining after the fibrin and suspended material (such as cells)have been depleted.

Blood samples, such as serum samples, can be obtained by any suitablemethod. In one embodiment, a trough serum/plasma is used and theconcentration range is 12-20 mg. Sweat samples can be obtained using,for example, a PharmChek® sweat patch from Sudormed. The PharmChek®sweat patch includes a semi-occlusive dressing containing a medicalgrade cellulose blotter paper collection pad, covered by a thin layer ofpolyurethane and acrylate adhesives. At the end of the wear period, thepad is eluted with a suitable buffer, such as 2.5 mL of 0.2 M acetatebuffer with methanol at pH 5.0 (25:75) or with acetonitrile.Furthermore, the biological samples may also be tissue samples, whichare extracted into liquid medium for immunoassay. For example, hairsamples can be tested by extracting into a liquid medium. The samplesmay be diluted or modified to facilitate the assay.

The samples may be experimental samples generated by any chemical orbiological method. For example, the samples may be standards containingknown concentrations of levetiracetam or other substances used for assaycalibration.

In some embodiments, the biological sample will be diluted in a suitablesolution prior to assaying. In general, a solution suitable for dilutinga biological sample will include a buffer, such as phosphate bufferedsaline (PBS), and may include additional items, such as for example, anon-specific blocking agent, such as bovine serum albumin (BSA), adetergent, such as Triton-X-100, and the like.

Where desired, appropriate control samples for the assay include blood,serum, or urine collected from human subjects who have not receivedlevetiracetam (i.e., a negative control), or samples which contain aknown, predetermined amount of a levetiracetam analyte (i.e., a positivecontrol). Alternatively, test results can be compared to detectablesignal levels known to be associated with the presence or absence oflevetiracetam and/or correlated with an amount of levetiracetam, e.g., aserum level of levetiracetam.

The assays may optionally include use of a calibration standard.“Calibration standard”, as used herein, refers to an aqueous mediumcontaining levetiracetam at a predetermined concentration. In a certainembodiment, a series of these calibration standards are available at aseries of predetermined concentrations. In a certain embodiment, thecalibration standard is stable at ambient temperature. In a certainembodiment, the calibration standards are in a synthetic matrix. In acertain embodiment, the calibration standards are in a non-syntheticmatrix such as human serum.

In many embodiments, a suitable initial source for the human sample is ablood sample. As such, the sample employed in the subject assays isgenerally a blood-derived sample. The blood derived sample may bederived form whole blood or a fraction thereof, e.g., serum, plasma,etc., where in some embodiments the sample is derived from blood allowedto clot and the serum separated and collected to be used to assay.

In embodiments in which the sample is a serum or serum derived sample,the sample is generally a fluid sample. Any convenient methodology forproducing a fluid serum sample may be employed. In many embodiments, themethod employs drawing venous blood by skin puncture (e.g., fingerstick, venipuncture) into a clotting or serum separator tube, allowingthe blood to clot, and centrifuging the serum away from the clottedblood. The serum is then collected and stored until assayed. Once thepatient derived sample is obtained, the sample is assayed to determinethe level of levetiracetam analyte.

Immunoassay Reagents

Immunoassay reagents that find use alone or in combination in the assaysdescribed herein include anti-levetiracetam antibodies, levetiracetamconjugates, and levetiracetam (e.g., as a control or in competitivebinding assays). Immunoassay reagents can be provided in a bufferedaqueous solution. Such solutions may include additional components suchas surface active additives, organic solvents, defoamers, buffers,surfactants, and anti-microbial agents. Surface active additives areintroduced to maintain hydrophobic or low-solubility compounds insolution, and stabilize components in the solution. Examples includebulking agents such as betalactoglobulin (BLG) or polyethyleneglycol(PEG); defoamers and surfactants such as Tween-20, Plurafac A38, TritonX-100, Pluronic 25R2, rabbit serum albumin (RSA), bovine serum albumin(BSA), and carbohydrates. Examples of organic solvents can includemethanol and other alcohols. Various buffers may be used to maintain thepH of the solution during storage. Illustrative buffers include HEPES,borate, phosphate, carbonate, tris, barbital and the like.Anti-microbial agents also extend the storage life of the immunoassayreagent.

Anti-Levetiracetam Antibodies

Immunoassays generally involve at least one anti-levetiracetam antibody,which may be produced by the methods disclosed herein. In an embodiment,the assays involve using an antibody raised against a levetiracetamderivative-protein conjugate, particularly a low cross-reactivity withnon-levetiracetam molecules (i.e., molecules that are not levetiracetamor contain a levetiracetam moiety, such as present in a compound of thepresent disclosure, see, e.g., the compounds of Formulae 1-3) that maybe present in a reaction mixture. Anti-levetiracetam antibodies can bepolyclonal or monoclonal, more commonly monoclonal, antibodies, capableof specifically binding levetiracetam.

Depending upon the assay format, the anti-levetiracetam antibody can beoptionally detectably labeled, may be used in conjunction with asecondary antibody (i.e., an antibody that specifically binds ananti-levetiracetam antibody) that may be detectably labeled. Certaindetectable labels for antibodies are described infra.

Levetiracetam Conjugates

Levetiracetam conjugates variously find use as immunoassay reagentsdepending on the assay format. For example, levetiracetam conjugate canact as based on competitive binding reagent in competitive bindingassays, or can provide for a detectable signal when not bound by ananti-levetiracetam antibody (e.g., where the levetiracetam conjugate isa levetiracetam G6PDH conjugate). Certain levetiracetam conjugatesuseful as immunoassay reagents are described below.

Detectable Labels

A variety of detectably labels can be used in connection with thelevetiracetam conjugate assay reagents for use in the methods disclosedherein. Such detectable labels can be isotopic labels. In otherembodiments, the detectable labels are non-isotopic signal-generatingmoieties, such as fluorophores and enzymes. Certain detectable labelsare described below. It will be apparent that while the detectablelabels are described below in the context of their use in levetiracetamconjugates, many can also be adapted for use with anti-levetiracetamantibodies.

Fluorophores

“Fluorophore” as used herein refers to moiety that itself fluoresces,can be made to fluoresce, or can provide for quenching of fluorescenceof a fluorophore of a FRET pair (e.g., as in a FRET pair). In principle,any fluorophore can be used in the assays of the embodiments. Ingeneral, the fluorophore is selected so as to be compatible for use inthe assay format desired, and selected so as to be relativelyinsensitive to the assay conditions, e.g., pH, polarity, temperature andionic strength.

Certain fluorophores can be characterized as having the followingcharacteristics: a. A fluorescence lifetime of greater than about 15nsec; b. An excitation wavelength of greater than about 350 nm; c. AStokes shift (a shift to lower wave-length of the emission relative toabsorption) of greater than about 20 nm; d. For homogeneous assaysdescribed below, fluorescence lifetime should vary with binding status;and e. The absorptivity and quantum yield of the fluorophore should behigh. The longer lifetime is advantageous because it is easier tomeasure and more easily distinguishable from the Raleigh scattering(background). Excitation wavelengths greater than 350 nm reducebackground interference because most fluorescent substances responsiblefor background fluorescence in biological samples are excited below 350nm. A greater Stokes shift also allows for less background interference.

The fluorophores generally have a functional group available forconjugation either directly or indirectly to a levetiracetamintermediate to generate a levetiracetam conjugate having the attachedfluorophore.

Fluorophores for use in heterogeneous assays can be relativelyinsensitive to binding status. In contrast, fluorophores for use inhomogeneous assay can be sensitive to binding status, i.e., thefluorescence lifetime must be alterable by binding so that bound andfree forms can be distinguished.

Examples of fluorophores are naphthalene derivatives (e.g. dansylchloride), anthracene derivatives (e.g. N-hydroxysuccinimide ester ofanthracene propionate), pyrene derivatives (e.g. N-hydroxysuccinimideester of pyrene butyrate), fluorescein derivatives (e.g. fluoresceinisothiocyanate), rhodamine derivatives (e.g. rhodamine isothiocyanate),phycoerythin, and Texas Red.

Enzymes

In a certain embodiment, the signal-generating moiety is an enzyme. Fromthe standpoint of operability, a very wide variety of enzymes can beused. But, as a practical matter, some enzymes have characteristicswhich make them more readily adaptable to the methods disclosed herein.

The enzyme can be selected so as to be stable to provide for desirableshelf-life, e.g., stable when stored for a period of at least threemonths or at least six months at temperatures which are convenient tostore in the laboratory, normally −20° C. or above. The enzyme can beselected so as to have a satisfactory turnover rate at or near the pHoptimum for binding to the antibody, this is normally at about pH 6-10,usually 6.0 to 8.0. A product of the enzymatic reaction facilitated bythe enzyme can be either formed or destroyed as a result of the enzymereaction, and can provide a enzyme reaction product which absorbs lightin the ultraviolet region or the visible region, that is the range ofabout 250-750 nm., usually 300-600 nm. The enzyme may also have asubstrate (including cofactors) which has a molecular weight in excessof 300, or in excess of 500. The enzyme which is employed or otherenzymes, with like activity, will not be present in the sample to bemeasured, or can be easily removed or deactivated prior to the additionof the assay reagents. Also, the enzyme can be selected so as to avoidthe impact of any naturally occurring inhibitors for the enzyme that maybe present in samples to be assayed or as some other component of thereaction mixture.

Although enzymes of up to 600,000 molecular weight can be employed,usually relatively low molecular weight enzymes will be employed of from10,000 to 300,000 molecular weight, more usually from about 10,000 to150,000 molecular weight, and frequently from 10,000 to 100,000molecular weight. Where an enzyme has a plurality of subunits themolecular weight limitations refer to the enzyme and not to thesubunits.

It may be desirable to select an enzyme that is susceptible todetectable labeling. In this instance, the enzyme can be detectablelabeled using appropriate detectable labels exemplified herein. Certainenzymes include, but are not limited to: alkaline phosphatase,horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase,lactate dehydrogenase, β-galactosidase, and urease. Also, a geneticallyengineered fragment of an enzyme may be used, such as the donor andacceptor fragment of β-galactosidase utilized in CEDIA immunoassays(see, e.g., Henderson D R et al. Clin Chem. 32(9):1637-1641 (1986));U.S. Pat. No. 4,708,929. These and other enzymes which can be used havebeen discussed in detail by Eva Engvall in Enzyme Immunoassay ELISA andEMIT in Methods in Enzymology, 70:419-439 (1980) and in U.S. Pat. No.4,857,453.

In a certain embodiment, the enzyme is glucose-6-phosphate dehydrogenase(G6PDH) and it is attached to a levetiracetam derivative, thus forming alevetiracetam-reactive partner conjugate.

An anti-levetiracetam antibody used in conjunction with suchlevetiracetam conjugates can be selected so as to specifically bind thelevetiracetam epitope presented by the levetiracetam enzyme conjugate,and thus affect activity of the levetiracetam enzyme conjugate.

For assays employing levetiracetam-enzyme conjugates, as a certainprotein conjugate, in which a levetiracetam derivative is labeled withan enzyme, the levetiracetam derivative can be attached to the enzyme byany suitable method. In certain embodiments, the chemistry describedherein for formation of immunogenic protein conjugates of levetiracetamderivatives is also used to prepare the enzyme conjugate. In this way,the levetiracetam moiety presented to the antibody can more mirror thelevetiracetam epitope to which the antibody specifically binds.

The selection procedure is exemplified using a levetiracetam-reactivepartner conjugate comprising G6PDH as the reactive partner and alevetiracetam derivative as the hapten. The first step in selecting anantibody involves testing the magnitude of antibody inhibition of alevetiracetam-reactive partner conjugate. In this step, the goal is todetermine and select for those antibodies which significantly inhibitthe enzyme activity of G6PDH. Antibodies which perform well in the firsttest are then subjected to a second test. Here, the antibody is firstincubated with levetiracetam. Next the levetiracetam-reactive partnerconjugate is added. A certain antibody preferentially binds tolevetiracetam instead of the levetiracetam-reactive partner conjugate.The reduction in binding to the levetiracetam-reactive partner conjugatewould be visible as an increase G6PDH activity.

Detection

Via Fluorescence

When a fluorescently labeled analyte (i.e., levetiracetam antigen orantibody) is employed, the fluorescence emitted is proportional (eitherdirectly or inversely) to the amount of analyte. The amount offluorescence is determined by the amplitude of the fluorescence decaycurve for the fluorescent species. This amplitude parameter is directlyproportional to the amount of fluorescent species and accordingly to theanalyte.

In general spectroscopic measurement of fluorescence is accomplished by:a. exciting the fluorophore with a pulse of light; b. detecting andstoring an image of the excitation pulse and an image of all thefluorescence (the fluorescent transient) induced by the excitationpulse; c. digitizing the image; d. calculating the true fluorescenttransient from the digitized data; e. determining the amplitude of thefluorescent transient as an indication of the amount of fluorescentspecies.

According to the method, substantially all of the fluorescence emittedby the fluorescent species reaching the detector as a function of timefrom the instant of excitation is measured. As a consequence, the signalbeing detected is a superimposition of several component signals (forexample, background and one analyte specific signal). As mentioned, theindividual contributions to the overall fluorescence reaching thedetector are distinguished based on the different fluorescence decayrates (lifetimes) of signal components. In order to quantitate themagnitude of each contribution, the detected signal data is processed toobtain the amplitude of each component. The amplitude of each componentsignal is proportional to the concentration of the fluorescent species.

Via Enzyme

Detection of the amount of product produced by thelevetiracetam-reactive partner conjugate of the embodiments can beaccomplished by several methods which are known to those of skill in theart. Among these methods are colorimetry, fluorescence, andspectrophotometry. These methods of detection are discussed in“Analytical Biochemistry” by David Holme, Addison-Wesley, 1998, which isincorporated herein by reference.

Solid Supports

The levetiracetam conjugates and/or the anti-levetiracetam antibodies tobe used as reagents in an assay can be insolubilized by attachment to asolid phase. This can be, for example, a wall of a vessel containing thereagent, to a particulate, or to a large molecular weight carrier thatcan be kept in suspension but is removable by physicochemical means,such as centrifugation or microfiltration. The attachment need not becovalent, but is at least of sufficient permanence to withstand anyseparation techniques (including washes) that are part of the assayprocedure. Certain particulate materials include agarose, polystyrene,cellulose, polyacrylamide, latex particles, magnetic particles, andfixed red cells. Examples of commercially available matrices includeSepharose® (Pharmacia), Poros® resins (Roche Molecular Biochemicals),Actigel Superflow™ resins (Sterogene Bioseparations Inc.), andDynabeads™ (Dynal Inc.). The choice is not critical, and will generallydepend on such features as stability, capacity, accessibility of thecoupled antibody, flow rate (or the ability to disperse the resin in thereaction mixture), and ease of separation.

Assay Formats

As noted above, immunoassays for detection of levetiracetam can be of avariety of formats, In general, the immunoassays involve combining oneor more immunoassay reagents (e.g., at least a anti-levetiracetamantibody) with a test sample (i.e., a sample suspected of containinglevetiracetam) in a reaction mixture. “Reaction mixture” generallyrefers to the combination of a sample suspected of containinglevetiracetam and one or more immunoassay reagents as exemplified in thepresent disclosure to facilitate detection of the presence or absence oflevetiracetam in the sample, where the detection may be qualitative orquantitative. The reaction mixture is usually an aqueous solution,although the immunoassay reagent(s) may be in solution or immobilized ona support (e.g., a substrate such as a bead). The reaction mixture caninclude other components compatible with the immunoassay, e.g., buffers,and the like.

Immunoassays usually are classified in one of several ways. For example,immunoassays can be classified according to the mode of detection used,i.e., enzyme immunoassays, radio immunoassays, fluorescence polarizationimmunoassays, chemiluminescence immunoassays, turbidimetric assays, etc.Another grouping method is according to the assay procedure used, i.e.,competitive assay formats, sandwich-type assay formats as well as assaysbased on precipitation or agglutination principles. In the instantapplication, a further distinction is made depending on whether washingsteps are included in the procedure (so-called heterogeneous assays) orwhether reaction and detection are performed without a washing step(so-called homogeneous assays). Certain assays are described in moredetail below.

Homogeneous and Heterogeneous Immunoassays

Immunoassays may be described as heterogeneous or homogeneous.“Homogeneous immunoassay”, as used herein, refers to an assay methodwhere the complex is typically not separated from unreacted reactioncomponents, but instead the presence of the complex is detected by aproperty which at least one of the reactants acquires or loses as aresult of being incorporated into the complex. Homogeneous assays knownin the art include systems involving fluorochrome and fluorochromequenching pairs on different reagents; enzyme and enzyme inhibitor pairson different reagents; chromophore and chromophore modifier pairs ondifferent reagents; and latex agglutination assays.

A certain homogeneous assay is the quantitative homogeneous enzymeimmunoassay in which a levetiracetam moiety is conjugated to an activeenzyme. The conjugation is arranged so that the binding of ananti-levetiracetam antibody to the derivative affects enzymatic activityin a qualitative or quantitative fashion. If a sample containinglevetiracetam is premixed with the antibody, the antibody will complexwith the levetiracetam and be prevented from binding to the enzymeconjugate. In this way, the activity of the enzyme can be correlatedwith the amount of levetiracetam present in the sample.

G6PDH is a certain enzyme useful in such assays. In one embodiment, theG6PDH is a variant of a naturally-occurring G6PDH in which one or morelysine residues are deleted or substituted, or one or more cysteineresidues are introduced. For example, Leuconostoc mesenteroides G6PDHare dimeric enzymes that have the ability to catalyze the oxidation ofD-glucose-6-phosphate to D-glucono-delta-lactone-6-phosphate byutilizing either NAD⁺ or NADP⁺. This property of using NAD⁺differentiates these enzymes from human G6PDH, which utilizes only NADP⁺effectively, and allows L. mesenteroides-specific G6PDH activity to bemeasured in the presence of human G6PDH, as for example in humansamples. G6PDHs from L. mesenteroides are used in current EMIT™homogeneous immunoassays (Syva Company, Palo Alto, Calif., U.S.A.). Twocertain genera of bacteria from which to select G6PDH are Leuconostocand Zymomonas. Within these genera L. mesenteroides, L. citreum, L.lactis, L. dextranicum, and Z. mobilis are of most interest, L.mesenteroides, L. citreum, L. lactis are specific examples.

Another example of a homogeneous assay system is the cloned enzyme donorimmunoassay.

In a separation-based or “heterogeneous” assay, the detecting of acomplex of an anti-levetiracetam antibody and an analyte involves aprocess wherein the complex formed is physically separated from eitherunreacted analyte, unreacted antibody, or both.

In a heterogeneous immunoassay, a complex of an anti-levetiracetamantibody and an analyte may be first formed in the fluid phase, and thensubsequently captured by a solid phase reagent or separated on the basisof an altered physical or chemical property, such as by gel filtrationor precipitation. Alternatively, one of the reagents may be attached toa solid phase before contacting with other reagents, and then thecomplex may be recovered by washing the solid phase free of unreactedreagents. Separation-based assays typically involve use of a labeledderivative or antibody to facilitate detection or quantitation of thecomplex. Suitable labels include radioisotopes such as ¹²⁵I, enzymessuch as peroxidase and β-galactosidase, and fluorescent labels such asfluorescein isothiocyanate. The separation step involves removinglabeled reagent present in complex form from unreacted labeled reagent.The amount of label in the complex can be measured directly or inferredfrom the amount left unreacted.

Sandwich and Competition Assays

Assays of this disclosure include both sandwich and competition assays.Sandwich assays typically involve forming a complex in which the analyteto be measured is sandwiched between one reagent, such as a firstantibody used ultimately for separation of the complex, and anotherreagent, such as a second antibody used as a marker for the separatedcomplex. Competition assays involve a system in which the analyte to bemeasured competes with an derivative of the analyte for binding toanother reagent, such as an antibody. An example of a competition assayusing EMIT® is described in U.S. Pat. No. 3,817,837.

In one embodiment, the immunoassay further comprises adding alevetiracetam conjugate comprising a levetiracetam moiety and adetectable label to the sample. The presence or absence of levetiracetamin the sample can be detected by detecting the datable label. Thedetectable label may comprise an enzyme and the detecting is by assayingactivity of the enzyme. In an embodiment, the enzyme is a dehydrogenase,more particularly, G6PDH.

Lateral Flow Chromatography

The compounds and methods of the embodiments also encompass the use ofthese materials in lateral flow chromatography technologies. The essenceof lateral flow chromatography involves a membrane strip which comprisesa detection device, such as a non-isotopic signal generating moiety, forlevetiracetam. A sample from a patient is then applied to the membranestrip. The sample interacts with the detection device, producing aresult. The results can signify several things, including the absence ofthe levetiracetam in the sample, the presence of the levetiracetam inthe sample, and even the concentration of the levetiracetam in thesample.

A certain embodiment provides a method of qualitatively determining thepresence or absence of a levetiracetam in a sample, through the use oflateral flow chromatography. The basic design of the qualitative lateralflow device is as follows: 1) The sample pad is where the sample isapplied. The sample pad is treated with chemicals such as buffers orsalts, which, when redissolved, optimize the chemistry of the sample forreaction with the conjugate, test, and control reagents. 2) Conjugaterelease pad is typically a polyester or glass fiber material that istreated with a conjugate reagent such as an antibody colloidal goldconjugate. A typical process for treating a conjugate pad is to useimpregnation followed by drying. In use, the liquid sample added to thetest will redissolve the conjugate so that it will flow into themembrane. 3) The membrane substrate is usually made of nitrocellulose ora similar material whereby antibody capture components are immobilized.4) A wicking pad is used in tests where blood plasma must be separatedfrom whole blood. An impregnation process is usually used to treat thispad with reagents intended to condition the sample and promote cellseparation. 5) The absorbent pad acts as a reservoir for collectingfluids that have flowed through the device. 6) The above layers andmembrane system are laminated onto a plastic backing with adhesivematerial which serves as a structural member.

A certain embodiment provides a method of qualitatively determining thepresence of a levetiracetam in a sample, through the use of lateral flowchromatography. In this embodiment, the membrane strip comprises asample pad, which is a conjugate release pad (CRP) which comprises anantibody that is specific for the levetiracetam. This antibody isconjugated to a non-isotopic signal-generating moiety, such as acolloidal gold particle. Other detection moieties useful in a lateralflow chromatography environment include dyes, colored latex particles,fluorescently labeled latex particles, non-isotopic signal generatingmoieties, etc. The membrane strip further comprises a capture line, inwhich the levetiracetam derivative antigen is immobilized on the strip.In some embodiments, this immobilization is through covalent attachmentto the membrane strip, optionally through a linker. In otherembodiments, the immobilization is through non-covalent attachment tothe membrane strip. In still other embodiments, the immobilelevetiracetam derivative in the capture line is attached to a reactivepartner, such as an immunogenic carrier like BSA.

Sample from a patient is applied to the sample pad, where it can combinewith the antibody in the CRP, thus forming a solution. This solution isthen allowed to migrate chromatographically by capillary action acrossthe membrane. When the levetiracetam is present in the sample, alevetiracetam-antibody complex is formed, which migrates across themembrane by capillary action. When the solution reaches the captureline, the levetiracetam-antibody complex will compete with the immobilelevetiracetam for the limited binding sites of the antibody. When asufficient concentration of levetiracetam is present in the sample, itwill fill the limited antibody binding sites. This will prevent theformation of a colored antibody-immobile levetiracetam complex in thecapture line. Therefore, absence of color in the capture line indicatesthe presence of levetiracetam in the sample.

In the absence of levetiracetam in the sample, a coloredantibody-immobile levetiracetam complex will form once the solutionreaches the capture line of the membrane strip. The formation of thiscomplex in the capture line is evidence of the absence of levetiracetamtherapeutic in the sample.

A certain embodiment provides a method of quantitatively determining theamount of a levetiracetam in a sample, through the use of lateral flowchromatography. This technology is further described in U.S. Pat. Nos.4,391,904; 4,435,504; 4,959,324; 5,264,180; 5,340,539; and 5,416,000,among others, which are herein incorporated by reference. In oneembodiment, the antibody is immobilized along the entire length of themembrane strip. In general, if the membrane strip is made from paper,the antibody is covalently bound to the membrane strip. If the membranestrip is made from nitrocellulose, then the antibody can benon-covalently attached to the membrane strip through, for example,hydrophobic and electrostatic interactions. The membrane strip comprisesa CRP which comprises the levetiracetam attached to a detector moiety.In a certain embodiment, the detector moiety is an enzyme, such ashorseradish peroxidase (HRP).

Sample from a patient is applied to the membrane strip, where it cancombine with the levetiracetam/detector molecule in the CRP, thusforming a solution. This solution is then allowed to migratechromatographically by capillary action across the membrane. When thelevetiracetam is present in the sample, both the sample levetiracetamand the levetiracetam/detector molecule compete for the limited bindingsites of the antibody. When a sufficient concentration of levetiracetamis present in the sample, it will fill the limited antibody bindingsites. This will force the levetiracetam/detector molecule to continueto migrate in the membrane strip. The shorter the distance of migrationof the levetiracetam/detector molecule in the membrane strip, the lowerthe concentration of levetiracetam in the sample, and vice versa.

When the levetiracetam/detector molecule comprises an enzyme, the lengthof migration of the levetiracetam/detector molecule can be detected byapplying an enzyme substrate to the membrane strip. Detection of theproduct of the enzyme reaction is then utilized to determine theconcentration of the levetiracetam in the sample. In a certainembodiment, the enzyme's color producing substrate such as a modifiedN,N-dimethylaniline is immobilized to the membrane strip and3-methyl-2-benzothiazolinone hydrazone is passively applied to themembrane, thus alleviating the need for a separate reagent to visualizethe color producing reaction.

Fluorescence Polarization Immunoassay for Levetiracetam

Fluorescence polarization immunoassay (FPIA) technology is based uponcompetitive binding between an antigen/drug in a sample and a knownconcentration of labeled antigen/drug. FPIA technology is described in,for example, U.S. Pat. Nos. 4,593,089, 4,492,762, 4,668,640, and4,751,190, which are incorporated herein by reference. Accordingly, theFPIA reagents, systems, and equipment described in the incorporatedreferences can be used with anti-levetiracetam antibodies which are alsoanti-levetiracetam analog antibodies.

The FPIA technology can be used to identify the presence oflevetiracetam and can be used in assays that quantify the amount oflevetiracetam in a sample. In part, the rotational properties ofmolecules in solution allow for the degree of polarization to bedirectly proportional to the size of the molecule. Accordingly,polarization increases as molecular size increases. That is, whenlinearly polarized light is used to excite a fluorescent-labeled orother luminescent-labeled levetiracetam or derivative thereof, which issmall and rotates rapidly in solution, the emitted light issignificantly depolarized. When the fluorescent-labeled levetiracetam orderivative interacts with or is bound to an antibody, the rotation isslowed and the emitted light is highly polarized. This is because theantibody significantly and measurably increases the size of the complex.Also, increasing the amount of unlabeled levetiracetam in the sample canresult in decreased binding of the fluorescent-labeled levetiracetam orderivative by the anti-levetiracetam antibody, and thereby decrease thepolarization of light emitted from sample. The quantitative relationshipbetween polarization and concentration of the unlabeled levetiracetam inthe sample can be established by measuring the polarization values ofcalibrations with known concentrations of levetiracetam. Thus, FPIA canbe used to identify the presence and concentration of levetiracetam in asample.

In one embodiment, the assay involves an FPIA assay system. An exampleof components of the FPIA system can include the following: i)monoclonal or polyclonal anti-levetiracetam antibodies capable ofspecifically binding to levetiracetam and a levetiracetam derivative;ii) a sample suspected of containing the levetiracetam; and iii)levetiracetam derivative labeled with a fluorescent moiety, such asfluorescein. Alternatively, the system can be provided as a kitexclusive of the sample. Additionally, the system can include variousbuffer compositions, levetiracetam concentration gradient compositionsor a stock composition of levetiracetam, and the like.

Homogeneous Microparticle Immunoassay for Levetiracetam

Homogeneous microparticles immunoassay (“HMI”) technology, which can bereferred to as immunoturbidimetric assays, is based on the agglutinationof particles and compounds in solution. When particles and/or chemicalcompounds agglutinate, particle sizes can increase and increase theturbidity of a solution. Accordingly, anti-levetiracetam antibodies canbe used with microparticles and levetiracetam derivatives in order toassess the presence, and optionally the amount, of levetiracetam in asample. HMI technologies can be advantageous because the immunoassayscan be performed on blood, blood hemolysate, serum, plasma, tissue,and/or other samples. HMI assays can be configured to be performed withlevetiracetam and/or a levetiracetam derivative loaded onto amicroparticle, or with an anti-levetiracetam antibody loaded onto amicroparticle. HMI or immunoturbidimetric assays are well known in theart for measuring agglutination of substances in a sample.

Immunoturbidimetric assay technologies are described in, e.g., U.S. Pat.Nos. 5,571,728, 4,847,209, 6,514,770, and 6,248,597, which are includedherein by reference. Such assays involve light attenuation,nephelometric, or turbidimetric methods. The formation of anagglutinated compound AB from levetiracetam (A) and anti-levetiracetamantibody microparticle binding partner (B) can be measured by the changewhich occurs in the scattering or absorption of the incident lightdirected into the sample. Alternatively, the anti-levetiracetam antibody(A) can bind with a levetiracetam or derivative loaded microparticle.When suspendable particles having an immobilized binding partner areused, there is an enhancement of the effects, which makes it possible todetermine considerably lower levetiracetam concentrations. Thesehomogeneous methods can be carried out quickly and simply, and permit,in particular, the automation of sample analyses as described in moredetail below.

Cloned Enzyme Donor Immunoassays for Levetiracetam

Cloned enzyme donor Immunoassays (“CEDIA®”, Roche Diagnostics), as arebased upon the competition of levetiracetam in the biological samplewith a levetiracetam conjugate containing an inactive geneticallyengineered enzyme-donor (“ED”) fragment such as from β-D-galactosidegalactohydrolase or β-galactosidase (“β-gal”) from E. coli, for bindingto an antibody capable of binding levetiracetam. If levetiracetam ispresent in the sample it binds to the antibody, leaving the ED portionof the ED-derivative conjugate free to restore enzyme activity ofβ-D-galactoside galactohydrolase or B gal in the reaction mixture so asto be capable of association with enzyme acceptor (“EA”) fragments. Theactive enzyme comprised of the ED and EA is then capable of producing aquantifiable reaction product when exposed to an appropriate substrate.A preferred substrate is chlorophenol red-β-D-galactopyranoside(“CPRG”), which can be cleaved by the active enzyme into galactose andCPR, wherein CPR is measured by absorbency at about wavelength 570 nm.In the instance levetiracetam is not present in the sample, the antibodybinds to the ED-derivative conjugate, thereby inhibiting association ofthe ED fragments with the EA fragments and inhibiting restoration ofenzyme activity. The amount of reaction product and resultant absorbancechange are proportional to the amount of levetiracetam in the sample.

Chemiluminescent Heterogeneous Immunoassays for Levetiracetam

A competitive assay using chemiluminescent microparticle immunoassay(“CMIA”) technology can also be used to assess whether or notlevetiracetam is present in a sample. Various types of CMIA technologiesare well known in the art of heterogeneous immunoassays for determiningthe presence and/or amount of a chemical entity in a sample. CMIA assayscan include the use of anti-levetiracetam antibodies, which are capableof binding to levetiracetam and its derivatives, which are coupled toparticles, such as magnetic particles or particles suitable forseparation by filtration, sedimentation, and/or other means.Additionally, a tracer, which can include a levetiracetam derivativelinked to a suitable chemiluminescent moiety, can be used to competewith free levetiracetam in the patient's sample for the limited amountof anti-levetiracetam antibody on the particle. After the sample,tracer, and antibody particles interact and a routine wash step hasremoved unbound tracer, the amount of tracer bound to antibody particlescan be measured by chemiluminescence, wherein chemiluminescence isexpressed in Relative Light Units (RULE). The amount ofchemiluminescence is inversely related to the amount of free drug in thepatient's sample and concentration is determined by constructing astandard curve using known values of the drug.

Other Immunoassays for Levetiracetam

The levetiracetam derivatives, conjugates, antibodies, immunogens,and/or other conjugates described herein are also suitable for any of anumber of other heterogeneous immunoassays with a range of detectionsystems including but not limited to enzymatic or fluorescent, and/orhomogeneous immunoassays including but not limited to rapid lateral flowassays, and antibody arrays, as well as formats yet to be developed.

While various immunodiagnostic assays have been described herein thatutilize the levetiracetam derivatives, conjugates, antibodies,immunogens and/or tracers, such assays can also be modified as is wellknown in the art. As such, various modifications of steps or acts forperforming such immunoassays can be made within the scope of theembodiments.

Kits

The present disclosure also provides kits that find use in practicingthe subject methods, as described above. The kits of the embodiments cancomprise an anti-levetiracetam antibody in a container, and may comprisea levetiracetam conjugate (e.g., for use in a competitive binding assay,for use in an enzyme-based assay, and the like). The kits may alsoinclude a calibration standard and/or control standard useful inperforming the assay; and, optionally, instructions on the use of thekit. Kit components can be in a liquid reagent form, a lyophilized form,or attached to a solid support. The reagents may each be in separatecontainers, or various reagents can be combined in one or morecontainers depending on cross-reactivity and stability of the reagents.The sample, suspected of containing a levetiracetam, and a calibrationmaterial, containing a known concentration of the levetiracetam, areassayed under similar conditions. Levetiracetam concentration is thencalculated by comparing the results obtained for the unknown specimenwith results obtained for the standard. This is commonly done byconstructing a calibration or dose response curve.

Various ancillary materials may be employed in an assay in accordancewith the embodiments. In a certain embodiment, buffers and/orstabilizers are present in the kit components. In a certain embodiment,the kits comprise indicator solutions or indicator “dipsticks”,blotters, culture media, cuvettes, and the like. In a certainembodiment, the kits comprise indicator cartridges (where a kitcomponent is bound to a solid support) for use in an automated detector.In a certain embodiment, additional proteins, such as albumin, orsurfactants, particularly non-ionic surfactants, may be included. In acertain embodiment, the kits comprise an instruction manual that teachesa method of the embodiments and/or describes the use of the componentsof the kit. Reagents and buffers used in the assays can be packagedseparately or in combination into kit form to facilitate distribution.The reagents are provided in suitable containers, and typically providedin a package along with written instructions relating to assayprocedures.

An embodiment of the present disclosure relates to a kit forconveniently determining the presence or the absence of levetiracetam ina sample. The kit may comprise an anti-levetiracetam antibody and alevetiracetam calibration standard. The levetiracetam calibrationstandard may comprise calibration and control standards useful inperforming the assay. The kits can also optionally comprise a conjugatecomprising a levetiracetam moiety and a detectable signal. In a certainembodiment, a detectable signal of the conjugate is an enzyme. In yetanother embodiment, the enzyme is glucose-6-phosphate dehydrogenase(G6PDH). In one embodiment, the G6PDH is a variant of anaturally-occurring G6PDH in which one or more lysine residues aredeleted or substituted, or one or more cysteine residues are introduced.

EXAMPLES

The following examples are offered by way of illustration and not by wayof limitation. It should be apparent that the invention can includeadditional embodiments not illustrated by example. Additionally, many ofthe examples have been performed with experimental protocols well knownin the art using the levetiracetam derivatives, antigens, immunogens,and anti-levetiracetam derivative antibodies prepared in accordance withthe present invention.

Example 1 Scheme 1 is a Schematic Representation of a Chemical ReactionShowing the Reaction Between (S)-2-aminobutyramide (3) and MethylItaconate (4) for Synthesizing(2S)-2-[4-(aminomethyl)-2-oxo-1-pyrrolidinyl]-butanamide HCl Derivative(11) of Levetiracetam Based on Benoit et al., J. Med. Chem. 2004, 47,530-549

Transformation of the ester function of (5), into the mesylate (9) usingstandard conditions allows the introduction of azide functionality.Hydrogenolysis of the azide (10) affords the aminomethyl derivative(11). Oxidation of (6) with KMnO₂ affords compound (7). Treatingcompound (5) with NaOH and MeOH then HCl affords compound (8).

Example 2 Scheme 2 is a Schematic Representation of a Chemical ReactionShowing the Reaction Between (S)-2-aminobutyramide (3) and Compound (12)Giving 1-((1S)-1-Carbamoylpropyl)-2-oxopyrrolidine-3-carboxylic Acid(13) and (2S)-2-(3-Hydroxymethyl-2-oxopyrrolidin-1-yl)butyramide (14)Followed by Oxidation to Give (15) Based on (Benoit et al., J. Med.Chem. 2004, 47, 530-549)

Using mechanical stirring, under inert atmosphere, a solution of6,6-dimethyl-5,7-dioxaspiro[2.5]octane-4,8-dione (12) and(2S)-2-aminobutanamide (3) in MeCN is warmed to 65° C. for 2 h. Anotherportion of (12) is added. After 3.5 h at 60° C., the reaction mixture isleft overnight at room temperature. The precipitate is filtered, isdissolved in water, and is purified on ion-exchange resin (AG50W-X4)with water, and the acidic fractions (pH <2) are collected to afford(13).

Using mechanical stirring, under inert atmosphere, CH₃I is addeddropwise to a suspension of K₂CO₃ and (13) in acetone at roomtemperature. The mixture is refluxed, and a second portion of CH₃I isadded dropwise. The reaction mixture is left overnight at roomtemperature, is refluxed, is cooled to room temperature, and isconcentrated in vacuo. The filtrate is extracted with AcOEt and isevaporated to dryness to afford a crude methyl ester which is usedwithout further purification. Using a magnetic stirrer, under inertatmosphere, NaBH₄ is added to a mixture of the crude methyl ester int-BuOH at room temperature. After heating at 80° C., the reactionmixture is cooled to room temperature and is quenched with water (1 l),and t-BuOH is removed in vacuo (bath temperature 30° C.). The aqueouslayer is saturated with NaCl, is lowered to pH 6.8, is extracted withCHCl₃-MeOH (80/20 (v/v)), and is concentrated to dryness to afford acrude alcohol (14). Treating compound (14) with KMnO₄, NaOH and H₂O canthen afford compound (15).

Example 3 Scheme 3 is a Schematic Representation of a Chemical ReactionDepicting the Acylation Reaction of Aminomethyl Derivative ofLevetiracetam (11) with Succinic Anhydride to Give Compound (16)

A solution of (11) in tetrahydrofuran (anhydrous) is combined withN,N-diisopropylethylamine (DIPEA), and is stirred under argon. Succinicanhydride and 4-dimethylaminopyridine (DMAP) are added to the abovesolution to form a reaction mixture. The reaction mixture is stirredunder argon for 12 hours, and the solvent is evaporated under reducedpressure to form a residue. The residue is purified by flash columnchromatography with ethyl acetate as an eluent. The fractions containingthe succinyl derivative (16) are combined and are concentrated to yielda final product.

Example 4 Scheme 4 is a Schematic Representation Depicting theThiolating Reaction for Introducing Sulphur into the AminomethylDerivative of Levetiracetam (11) Yielding Compound (18)

To a stirred solution of (11) in THF is added diisopropylethylamineN-succinimidyl-S-acetylthioacetate. The reaction mixture is stirred atroom temperature. TLC analysis of the mixture would show a new spot as aproduct in comparison with (11). The organic solvent is removed todryness by rotary evaporation under reduced pressure. The residue ispurified by flash column chromatography (silica gel) using ethylacetate/hexane (7/3) as an eluent to afford compound (17).

To a solution of (17) in degassed (N₂) MeOH and H₂O is added K₂CO₃ undernitrogen. The reaction mixture is stirred at room temperature undernitrogen for 1 hour. TLC analysis of the mixture would show thatstarting material (17) disappears and a new spot is formed as a product(silica gel, MeOH/CH₂Cl₂=1/9, I₂, Ellman's reagent). MeOH is filtered toremove excess K₂CO₃ and the filtrate is concentrated by rotaryevaporation in room temperature. The residue is dried under high vacuumfor 0.5 hour at room temperature to afford compound (18).

Example 5 Scheme 5 is a Schematic Representation of a Acylation ofLevetiracetam Derivative (11) with Bromoacetic N-Hydroxyl SuccinimideUnder Basic Condition to Give Compound (19)

To a solution of compound (11) in tetrahydrofuran (anhydrous) are addedN,N-diisopropylethylamine and a solution of bromoacetic N-hydroxylsuccinimide in tetrahydrofuran at 0° C. under argon. The reaction isstirred at room temperature. Water is added and most of tetrahydrofuranis removed by rotary evaporation. The aqueous phase is extracted withCH₂Cl₂. The combined organic phase is washed with water and is driedover MgSO4. The organic phase is filtered and is concentrated todryness. The residue is purified by flash column chromatography (silicagel) using ethyl acetate/hexane (2/3) as an eluent to afford compound(19).

Example 6 Scheme 6 is a Schematic Representation for the Synthesis ofCompound (25)

Rink amide resin (0.51 mequiv/g, 100-200 mesh) is placed in a glassvessel and is stirred in 20% v/v piperidine/DMF for 0.5 h. The resin isdrained and the entire deprotection is repeated. The resin is filtered,is washed (6×DMF), and is dried. The resin is suspended in DMF and istreated with9-fluorenylmethoxycarbonyl-N-ε-tert-butyloxycarbonyl-L-ornithine (19)followed by a solution of 1,3-diisopropyl carbodiimide and HOBt in DMF.The reaction mixture is stirred for 1 h at room temperature and then isfiltered and is washed (DMF), and the coupling process is repeated. Theresin is filtered, is washed (6×DMF, 6×CH₂Cl₂), is dried, and is used asit stands in the next steps (20). Amide resin is contained within afitted polypropylene syringe. Removal of the Fmoc group is achievedusing 20% piperidine in DMF to give compound (21). To the amino resin isadded 4-oxobutyric acid 4-methoxybenzyl ester in (MeO)₃CH. The resin isstirred and then is filtered, is washed with CH₂Cl₂, and is treated withsodium triacetoxyborohydride. The reaction mixture is stirred for 18 hat room temperature and then is filtered; is washed in the solventsequence MeOH, 3×CH₂Cl₂, 3×MeOH; and is dried (22). The N-Allocprotected amine is deprotected with Pd(PPh₃)₄, borohydride inDCM:MeOH:H₂O (5:4:1, v/v) is stirred at room temperature for 1 hour(23). To a suspension of resin (23) in tetrahydrofuran (anhydrous) isadded N,N-diisopropylethylamine and a solution of bromoacetic N-hydroxylsuccinimide in tetrahydrofuran at 0° C. under argon. The reactionmixture is stirred at room temperature for 2 hours. Water is added andmost of tetrahydrofuran is removed by rotary evaporation. The aqueousphase is extracted with CH₂Cl₂ (3×30 mL). The combined organic phase iswashed with water and is dried over MgSO₄. The organic phase is filteredand is concentrated to dryness. The residue is purified by flash columnchromatography (silica gel) using ethyl acetate/hexane (2/3) as aneluent to afford compound (24).

The resin is suspended in trifluoroacetic acid/H₂O (95/5) with 5% oftriisopropylsilane with vortex agitation and then is filtered and iswashed (CH₂Cl₂×2). The filtrate is concentrated and the residue isdissolved in CH₂Cl₂ and is concentrated once more. The desired compound(25) is purified.

Example 7 Scheme 7 is a Schematic Representation of Synthesis ofLevetiracetam Derivative (30)

Compound (30) can be synthesized similarly as described in Example 6using N-α-Fmoc-L-glutamic acid γ-t-butyl ester (26) as the startingmaterial.

Example 8 Scheme 8 is a Schematic Representation of Synthesis ofLevetiracetam Derivative (30)

Compound (31) can be synthesized similarly as described in Example 6using Fmoc-S-trityl-L-homocysteine (32) as the starting material.

Example 9 Scheme 9 is a Schematic Representation of a Chemical Reactionfor Converting a Levetiracetam Derivative (11) into an Immunogen(11-KLH)

Lyophilized succinylated KLH (33) (Sigma) is reconstituted withdeionized water. The KLH solution is dialyzed overnight with two changesMES buffer (0.1 M MES, 0.9 M NaCl, 0.02% NaN3, pH 4.7). After dialysis,succinylated KLH is transferred to a reaction vial. Compound (11) isdissolved in dry DMF and is added to the reaction vial slowly. EDC(Pierce) is dissolved in deionized water and is immediately added to KLHsolution. Additional EDC aliquots (10 per addition) are added untilslight precipitation occurred during the conjugation reaction. Thereaction is allowed to proceed for approximately 2 h under constantmixing at room temperature to give immunogen (11-KLH). The reactionmixture is then dialyzed against three changes of HEPES buffer (0.05 M,pH 7.2, 1 mM EDTA).

Example 10 Schemes 10A-10F are Schematic Representations for theSynthesis of Compound (44)

In a 10-L, three-necked flask fitted with mechanical stirrer andrefluxing condenser, under nitrogen atmosphere, 1,226 g (12 mol, 1equiv) of (2S)-2-aminobutanamide and 1,912 mL (2,150 g, 13.2 mol, 1.1equiv) of dimethyl itaconate is dissolved in 6.13 L of MeOH. The mixtureis refluxed for 10 h and cooled slowly to 20° C. over 4 h. It wasfiltered, the precipitate is washed with MeOH, and the combined organicphases are concentrated to dryness to give 3283 g of the crudeintermediate. In a 20-L, three-necked flask fitted with a mechanicalstirrer, Rashig column, and distillation arm, under inert atmosphere,the above obtained, crude intermediate and 84.7 g (891 mmol, 0.1 equiv)of 2-hydroxypyridine is dissolved in 11.6 L of toluene. The mixture isrefluxed and the methanol formed is distilled off for 8 h until 480 mLof methanol is collected. The mixture is cooled and concentrated todryness to give 2,187 g of crude amide ester as a mixture ofdiastereoisomers in a 57.5/42.5 ratio.

The two diastereoisomers is separated by preparative LC Chiral Phase(Chiralpak AD 100 500 mm, EtOH H₂O 99.9/0.1)-column, the eluate isconcentrated to dryness to give 968 g of the crude 1 (first elutedcompound). The crude (34) is recrystallized from 2 L of AcOEt to give676 g of pure compound (34).

In a 2-L three-necked flask fitted with mechanical stirrer and refluxcondenser, under inert atmosphere, a solution of 133 g (583 mmol, 1equiv) of compound (34) in 200 mL of EtOH is added to 300 mL of EtOH,and the mixture is cooled to 0° C. Solid NaBH4 (66.2 g, 1.74 mol, 12equiv) is then added by portions over 1.5 h, while the temperature ismaintained between 2 and 4° C. After 2 h, the temperature is raised to12° C. for 1 h and lowered again to 2-4° C. Then, 240 mL of a saturatedsolution of NH₄Cl is added dropwise over 1 h, followed by 120 mL ofacetone, and the mixture is left overnight at room temperature. Themixture is filtered and the precipitate washed with 370 mL of EtOH. Thecombined organic fractions are concentrated to dryness to give 148 g ofcrude product. It is suspended in 300 mL of CH₂Cl₂, stirred for 0.5 h,filtered, washed with 2100 mL of CH₂Cl, and dried to give 114 g ofcompound (6).

In a 4 L, three necked flask fitted with mechanical stirrer, droppingfunnel and refluxing condenser under inert atmosphere, 114 g (56 mmol, 1equiv) of compound (6) is dissolved in 2 L of CH₂Cl₂ and cooled to 0° C.Dry triethylamine (158.5 mL, 115 g, 2 equiv) is added in one portion,followed by dropwise addition of a solution o 66.3 mL (96.2 g, 1.5equiv) of methanesulfonyl chloride in 19 mL of CH₂Cl₂ over 1 h, whilethe temperature is maintained below 4° C. After 4 h, 7.5 mL ofmethanesulfonyl chloride and 15 mL of triethylamine are added, and themixture is kept overnight in the refrigerator. The mixture is filtered,the residue is washed with CH₂Cl₂, and the combined organic phases areconcentrated to dryness to give 216 g crude compound (9).

To a stirred solution of compound (9) (1.128 mg, 5 mmol) was addedsodium cyanide (0.6 g, 10 mmol) in DMSO (20 ml). The mixture solutionwas stirred at room temperature for 1 h and then to 100° C. for 3 h. Theorganic phase was filtered and the filtrate was concentrated underreduced pressure gave crude (35) as a white solid (0.3 g, 50% yield).

To a solution of (35) (8.29 g; 21.20 mmol) and cobaltous chloridehexahydrate (10.08 g 42.40 mmol) in 99% MeOH (350 mL) and THF (120 mL),was added NaBH₄ (8.04 g; 212 mmol) in portions. Evolution of hydrogengas was observed as well as the formation of a black precipitate. Whenthe addition was complete, stirring was continued for 2 h at roomtemperature. HCl (2 M, 70 mL) was then poured in the reaction mixture inorder to dissolve the black precipitate. After removal of MeOH and THF,the aqueous layer was made alkaline by the addition of concentratedNH₄OH and then extracted with diethyl ether (4×200 mL). The combinedextracts were washed with saturated sodium chloride solution, dried(MgSO₄), filtered and evaporated in vacuo. The crude product waschromatographed on a 4.5×30 cm column of silica gel gave pure (36) as awhite solid (4.46 g).

To a stirred solution of compound (36) (464 mg, 1 mmol) was added2-bromoacetyl bromide (200 mg, 1 mmol) in DCM (30 ml). The mixturesolution was stirred at room temperature for 1 h and then water wasadded to the solution. The organic phase was dried with Na₂SO₄. Theorganic phase was filtered and the filtrate was concentrated underreduced pressure gave crude (44) as a yellow solid (460 mg, 0.8 mmol,80% yield).

Example 11 Scheme 11 is a Schematic Representation of a ChemicalReaction for Converting a Levetiracetam Analog Ester (13) into KLHImmunogen (12-KLH)

To a stirred solution of (12) in dried DMF is added1-ethyl-3-(3-dimethylamino propyl)carbodiimide (EDAC) andN-hydroxysuccinimide (NHS) at ice bath temperatures. The mixture isstirred overnight to form compound (13). Ester formation is monitored byTLC analysis.

Two vials of lyophilized KLH (Pierce) is reconstituted with deionizedwater each and are pooled. The mixture is allowed to stand overnight at4° C. A buffer exchange is done by dialyzing overnight the KLH solutionagainst sodium bicarbonate buffer (0.1 M, pH 8.9). An aliquot of the KLHpreparation is transferred into a reaction vial. The solution of (13) isslowly added (10-20 μL per addition) to the solution of KLH (34) over aperiod of 2 h at ice bath temperatures. After the addition is completed,the mixture is stirred in a 4° C. cold room overnight. This solution isthen dialyzed against three changes of HEPES buffer (10 mM, pH 7.0, 1mM) gives immunogen (12-KLH).

Haptens (7), (8), (16), and (30) are conjugated with KLH using aconjugation procedure similar to that described above.

Example 12 Scheme 12 is a Schematic Representation of a ChemicalReaction for Converting a Levetiracetam Derivative Ester (13) intoGlucose-6-Phosphate Dehydrogenase Conjugate (12-G6PDH)

Lyophilized G6PDH (Worthington Biochem. Corp.) is reconstituted withdeionized water. The mixture is allowed to stand overnight at 4° C. Themixture is then dialyzed overnight at 4° C. against sodium bicarbonatebuffer (0.1 M, pH 8.9). After dialysis, the enzyme solution istransferred to a reaction vial.

Activated product compound (13) is added in 5 to 10 μL quantities to asolution of glucose-6-phosphate dehydrogenase (G6PDH, 0.1 M in sodiumcarbonate buffer) glucose-6-phosphate (G6P, 4.5 mg/mg G6PDH), and NADH(9 mg/mg G6PDH) in a pH 8.9 sodium carbonate buffer at ice bathtemperature. After the addition of each portion of solution of compound(13) a 2 μL aliquot is taken and is diluted 1:500 with enzyme buffer. A3 μL aliquot of this diluted conjugation mixture is assayed forenzymatic activity similar to that described in Example 17 below. Thereaction is monitored and is stopped at approximately 65% deactivationof enzyme activity. The mixture is desalted with a PD-10 pre-packedSephadex G-25 (Pharmacia, Inc.) and pre-equilibrated with HEPES buffer(10 mM, pH 7.0, 1 mM EDTA). The reaction mixture is applied to thecolumn and the protein fractions pooled. The pooled fractions aredialyzed against three (1.0 L each) changes of HEPES (10 mM, pH 7.0, 1mM EDTA) to yield a solution of conjugate (12-G6PDH).

Haptens (7), (8), (16), and (30) are conjugated with G6PDH using aconjugation procedure similar to that described above.

Example 13 Scheme 13 is a Schematic Representation of a ChemicalReaction for Converting KLH (34) into Bromoacetyl-KLH (36)

To a solution of KLH (20 mg) in NaH₂PO₄—Na₂HPO₄ buffer (pH=8.0, 0.1M,2.0 mL) at 4° C. (ice-bath) was added a solution of bromoacetic acid NHSester (5.8 mg, 0.024 mmol) in DMF (0.2 mL). The pH value was maintainedat 8.0. The reaction mixture was stirred in the cold-room (4° C.) for 16hours. The mixture was purified by a Sephadex G-50 column, eluting withNaH₂PO₄—Na₂HPO₄ buffer (pH=7.00, 0.025 M). The eluted fractions from thecolumn were monitored by UV at 280 nm. A clean separation betweenbromoacetyl-KLH and the hapten was obtained. Fractions containing theproduct are pooled together (8.0 mL) and concentrated to 3.0 mL ofbromoacetyl-KLH (36) by an Amicon concentrator for the next reaction.

Example 14 Scheme 14 is a Schematic Representation of a ChemicalReaction for Converting a Levetiracetam (18) Analog into a KLH Immunogen(18-KLH)

To a solution of bromoacetyl-KLH (36) (pH=8.00) is added thelevetiracetam derivative (18) solution slowly at 4° C. under nitrogen.The pH value is maintained at 8.0. The reaction is stirred at 4° C.(cold room) for 16 hours. The reaction mixture is separated using aSephadex G-25 column equilibrated with NaH₂PO₄—Na₂HPO₄ buffer (pH=7.0,0.1 M). The UV detector at 280 nm can be used to monitor the elutedfractions from the column. A clean separation between KLH immunogen andthe hapten can be obtained. Fractions containing protein are pooled andare concentrated. The concentration of immunogen (18-KLH) is measured byusing BCA Protein Concentration Assay.

Hapten (32) is conjugated with KLH using a conjugation procedure similarto that described above.

Example 15 Scheme 15 is a Schematic Representation of a ChemicalReaction for Converting G6PDH (35) into Bromoacetyl Glucose-6-PhosphateDehydrogenase (37)

100 μL DMF was added to bromoacetic acid NHS (Sigma 3.06 mg, 12.97 μM)and stirred. A 2.0 mL (10 mg/mL) G6PDH solution was prepared in 0.025 Mphosphate carbonate buffer, pH 7.2 and adjusted to pH 8.5 with 0.4 Mcarbonate buffer. 45 mg disodium G6P and 90 mg NADH, was dissolved inthe G6PDH solution. Bromoacetic acid NHS was added to G6PDH solution at5 μL increments. Enzyme activity was measured on the HITACHI 917analyzer after each addition. Bromoacetic acid NHS was added untilapproximately 63.0% enzyme deactivation was obtained. G6PDH conjugationsolution was dialyzed with 3×4 liter portions of 0.01 M phosphate, pH7.2.

Example 16 Scheme 16 is a Schematic Representation of a ChemicalReaction for Converting a Levetiracetam (18) Analog into aGlucose-6-Phosphate Dehydrogenase Conjugate (18-G6PDH)

Bromoacetyl Glucose-6-Phosphate Dehydrogenase (37) is buffer exchangedwith 50 mM phosphate-1.0 mM EDTA, pH 7.25. A solution of the protein ismixed with a dithioerythreitol (25 mM final concentration in thephosphate-EDTA buffer) and mixture incubated at 4° C. for 16 hours. Theprotein solution is then buffer exchanged with 50 mM phosphate, 1.0 mMEDTA, 5 mM DTT, pH 7.25. The protein solution is mixed with 40 foldmolar excess of a DMF solution of levetiracetam derivative (18) andreaction mixture is stirred gently at 4° C. for 16 to 24 hours. Excess(18) is separated from the enzyme-hapten conjugate by passing thereaction mixture over a column of Sephadex G 50 in 50 mM phosphate, pH7.0. The column fractions containing the enzyme-hapten conjugate arepooled by measuring absorption at 280 nm which gave conjugate(18-G6PDH).

Hapten (32) is conjugated with G6PDH using a conjugation proceduresimilar to that described above.

Example 17 Scheme 17 is a Schematic Representation of a ChemicalReaction for Converting a Levetiracetam (19) Analog into aGlucose-6-Phosphate Dehydrogenase Conjugate (19-G6PDH)

The levetiracetam derivative hapten (19) is designed for proteinscontaining cysteine groups such as mutant G6PDH or introduction ofthiol-groups by chemical reactions. See, U.S. Pat. Nos. 6,455,288,6,090,567, 6,033,890, which are incorporated by reference in theirentireties. SH-G6PDH is buffer exchanged with 50 mM phosphate-1.0 mMEDTA, pH 7.25. A solution of the enzyme is mixed with a solution ofdithioerythreitol (0.5 M solution in the phosphate-EDTA buffer) andmixture is incubated at 4° C. for 16 hours. The protein solution is thenbuffer exchanged with 50 mM phosphate-1.0 mM EDTA-0.025 mM DTT, pH 7.25.Thiol content of the protein is determined by titration with a solutionof dithiodipyridine, and is reported as thiols per mole of the protein.The protein solution is mixed with 40 fold molar excess of a DMFsolution of hapten (19) and reaction mixture is stirred gently at 4° C.for 16-24 hours. Excess hapten (19) is separated from the enzyme-haptenconjugate by passing the reaction mixture over a column of Sephadex G 50in 50 mM phosphate, pH 7.0. The column fractions containing theenzyme-hapten conjugate is pooled by measuring absorption at 280 nm togive conjugate (19-G6PDH).

Hapten (25) is conjugated with G6PDH using a conjugation proceduresimilar to that described above.

Example 18 Scheme 18 is a Schematic Representation of a ChemicalReaction for Converting a Levetiracetam (19) Analog into a KLH Immunogen(19-KLH)

The levetiracetam derivative hapten (19) is conjugated to proteins wherethiol groups are chemically introduced. Commercially available linker,N-succinimidyl-S-acetylthioacetate (40) is reacted with primary amine ofKLH (34), which adds protected sulfhydryls. Deprotection of protectedsulfhydryls with hydroxyl amine produces a desired thiolated KLH (41).Conjugation of hapten (19) with thiolated KLH (41) results in immunogen(19-KLH).

Lyophilized KLH (Pierce) is reconstituted with phosphate buffer (0.1 M,0.15 M NaCl, 1 mM EDTA, pH 8.0). The KLH (34) solution is transferred toa reaction vial. Immediately before reaction,N-Succinimidyl-S-acetylthioacetate (40, SATA) is dissolved in DMSO(results in ˜55 mM solution). The SATA solution is combined with theprotein solution. The contents are mixed and reaction mixture isincubated at room temperature for at least 30 minutes. A Sephadex G-50column is equilibrated with two column volumes of buffer (0.1 Mphosphate, 0.15 M NaCl, pH 7.2-7.5). The reaction mixture is applied tocolumn. Fractions (500 μL) are collected immediately. The fractions thatcontain protein are identified by measuring absorbance at 280 nm.Protein fractions are pooled. Deacylation to generate a sulfhydryl foruse in cross-linking is accomplished adding 1 deacetylation solution(0.5 M Hydroxylamine, 25 mM EDTA in PBS, pH 7.2-7.5). Contents are mixedand reaction mixture is incubated for 2 hours at room temperature.Sephadex G-50 desalting column is used to purify the sulfhydryl-modifiedprotein from the hydroxylamine in the deacetylation solution. The pooledfractions are concentrated using Amicon concentrator.

Dithiothreitol (DTT, 1 mM) is added to thiolated KLH (28) to ensurereduction of disulfide bonds. The solution is allowed to mix overnightat 4° C. Bromoacetamido levetiracetam derivative hapten (19) isdissolved in 0.2 mL DMF. Levetiracetam derivative hapten (19) DMFsolution is added in 5 to 10 μL quantities to a solution of thiolatedKLH (41). The reaction is continued overnight at 4° C. This solution isdialyzed against three changes of HEPES buffer (10 mM, pH 7.0, 1 mMEDTA). This procedure yields immunogen (19-KLH).

Hapten (25) is conjugated with KLH using a conjugation procedure similarto that described above.

Example 19

KLH (41) was conjugated to hapten (44) as described in Example 18 aboveresulting in immunogen (44-KLH).

Example 20 Scheme 20 is a Schematic Representation of a ChemicalReaction for Converting a Levetiracetam (19) Analog into a G6PDHConjugate (19-G6PDH)

Hapten (25) is conjugated with G6PDH using a conjugation proceduresimilar to that described in Example 19.

Example 21

G6PDH (43) was conjugated to hapten (44) as described in Example 20above resulting in conjugate (44-G6PDH).

Example 22 Preparation of Levetiracetam Derivative Antibodies Reactiveto Levetiracetam.

The levetiracetam antibodies and enzyme conjugates prepared as describedin the above examples may be employed in assays for the detection oflevetiracetam. Either of the immunogens can be injected into a mouse,sheep or rabbit to raise antibody. Polyclonal sera from κ live rabbitswere prepared by injecting six animals with immunogen (44-KLH). Thisimmunogenic formulation comprises 200 μg of the immunogen for the firstimmunization and 100 μg for all subsequent immunizations. Regardless ofimmunogen amount, the formulation was then diluted to 1 mL with sterilesaline solution. This solution was then mixed thoroughly with 1 mL ofthe appropriate adjuvant: Freund's Complete Adjuvant for firstimmunization or Freund's Incomplete Adjuvant for subsequentimmunizations. The stable emulsion was subsequently injectedsubcutaneously with a 19×1½ needle into New Zealand white rabbits.Injections were made at 3-4 week intervals. Bleeds of the immunizedrabbits were taken from the central ear artery using a 19×1 needle.Blood was left to clot at 37° C. overnight, at which point the serum waspoured off and centrifuged. Finally, preservatives were added in orderto form the polyclonal antibody material. Rabbit polyclonal antibodiesto levetiracetam produced by the above procedure immunized withimmunogen (44-KLH) are designated as #12019, #12020, #12021, #12022,#12023, and #12024. Rabbit polyclonal antibody #12019 is used inexamples below.

Rabbit polyclonal antibody #12019-P8 was used to measure a substantialchange in enzyme activity, generate a calibration curve, and evaluateassay precision, accuracy and specificity. The antibody was added intothe antibody diluent to prepare the antibody reagent. The antibodyreagent consists of antibody as prepared above, buffer, stabilizers,preservatives, and the substrates for the enzyme conjugate nicotinamideadenine dinucleotide (NAM and glucose-6-phosphate. Enzyme conjugatecomprising compound (44-G6PDH) G6PDH was added into the conjugatereagent to prepare the enzyme conjugate reagent. The enzyme conjugatereagent comprises the conjugate, buffer, stabilizers and preservatives.Enzyme conjugate (44-G6PDH) was used with rabbit polyclonal antibody#12019-P8 in examples below. This technique is generally applicable toproduce polyclonal antibodies to levetiracetam derivatives and assesstheir utility.

Monoclonal antibodies may be prepared using standard hybridomaprocedures as described in detail (Kohler, G. et al., Nature 256:495-497 (1976); Hurrell, Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press, Boca Raton, Fla. (1982)). This hybridomatechnique is generally applicable to produce monoclonal antibodies tothe levetiracetam derivatives.

Example 23 Roche HITACHI 917 Clinical Chemistry Analyzer.

The levetiracetam derivative antibodies and enzyme conjugates may beadvantageously used in a homogeneous assay format to detectlevetiracetam in samples. An enzyme immunoassay or ARK Assay, which is ahomogeneous enzyme immunoassay experiment, was performed to testantibodies prepared as described in Example 22. The ARK Assay forlevetiracetam was conducted using a liquid, ready-to-use, two-reagentkit. A clinical chemistry analyzer useful to set up the assay is HITACHI917. The HITACHI 917 is an automated biochemistry analyzer used bymedical laboratories to process biological fluid specimens, such asurine, cerebrospinal fluid, and most commonly, blood. Manufactured byBoehringer Mannheim, the HITACHI 917 is a commonly used routine chemicalchemistry. Levetiracetam containing sample was incubated with antibodyreagent followed by the addition of the enzyme conjugate reagent. Theenzyme conjugate activity decreases upon binding to the antibody. Asillustrated in FIGS. 2a and 2b , the enzyme conjugate, which is notbound to the antibody, catalyzes the oxidation of glucose 6-phosphate(G6P). The oxidation of G6P is coupled with the reduction of NAD⁺ toNADH, which was measured at 340 nm. The change in the absorbance at 340nm was measured spectrophotometrically. The levetiracetam concentrationin a specimen was measured in terms of G6PDH activity. The increase inthe rate at 340 nm was due to the formation of NADH and is proportionalto the enzyme conjugate activity. An assay calibration curve wasgenerated using levetiracetam spiked into negative calibrator matrix.The assay rate increases with increasing the concentration of drug inthe sample.

Example 24 Calibration Curve

Levetiracetam was dissolved in methanol to give a stock solution of 1000μg/mL. Pooled human serum was aliquoted in 10 mL portions. Levetiracetamstock solution was added to the aliquots of human serum in preparing aseries of known concentrations of levetiracetam calibrators ranging fromκ to 100 μg/mL. Similarly, Quality Control samples were prepared (15.0,35.0 and 75.0 μg/mL). The antibody/substrate reagent #11341P4-6-Abcontaining antibody ##12019-P8 was assayed with enzyme reagent#110607-D63-E containing conjugate (44-G6PDH). Calibration curves weregenerated on the HITACHI 917 automated clinical chemistry analyzer, asdescribed in Example 23 by assaying each level in duplicate. An exampleof these calibrator rates is shown in Table 2 and the graph is shown inFIG. 3.

TABLE 2 Calibrator Reaction Rate Levetiracetam Conc. Reaction Rate(mA/min) (μg/mL) Average of Duplicates 0.0 522.6 5.0 547.4 10.0 561.225.0 586.9 50.0 607.5 100.0 626.0

Example 25 Accuracy of the Measurement.

Three levetiracetam Quality Control samples were prepared as describedin Example 24 to give concentrations of levetiracetam of 15.0, 35.0 and75.0 μg/mL. Enzyme Conjugate Reagent #110607-D63-E containing conjugate(44-G6PDH) and Antibody Reagent #11341P4-6-Ab containing antibody##12019-P8 was used to generate precision data shown in Table 3. Theaccuracy data were derived from 2 runs on the same day. Each run forgenerating a calibration curve includes 10 replicates of each QC levelper run with a total of 20 replicates from 2 runs. Quantification wasperformed on the HITACHI 917 analyzer as described in Example 23. Also,the accuracy of the measurement was calculated as a percentage of thenominal value of the QC samples (Table 3).

TABLE 3 Precision QC Conc. Mean Accuracy N (μg/mL) (μg/mL ± SD) (%) 1015.0 14.88 ± 1.02  99.21 10 35.0 35.77 ± 2.85 102.19 10 75.0 77.16 ±4.48 102.88

Example 26 Specificity of the Immunoassay in the Presence of2-Pyrrolidone-N-Butyric Acid, a Levetiracetam Metabolite (FIGS. 1(1) and1(2)).

The major metabolic pathway of levetiracetam (24% of dose) is anenzymatic hydrolysis of the acetamide group. The metabolites have noknown pharmacological activity and are renally excreted. To human serumnegative for levetiracetam was added levetiracetam dissolved in methanolto achieve a concentration of concentration of 30 μg/mL of levetiracetam(FIG. 1(1)).

To a second sample was added 2-pyrrolidone-N-butyric acid dissolved inmethanol to achieve a concentration of 150.0 μg/mL2-pyrrolidone-N-butyric acid (FIG. 1(2)). To a third sample was added 30μg/mL of levetiracetam plus 406.4 μg/mL 2-pyrrolidone-N-butyric acid(FIG. 1(2)). The samples were assayed on the HITACHI 917. Thelevetiracetam metabolite, 2-pyrrolidone-N-butyric acid was tested in theimmunoassay for potential crossreactivity with Enzyme Conjugate Reagent#110607-D63-E containing conjugate (44-G6PDH) and Antibody Reagent#11341P4-6-Ab containing antibody #12019-P8. As shown in Table 5,antibody #12019-P8 does not crossreact with 2-pyrrolidone-N-butyric acidmetabolite, indicating a highly specific antibody was produced.

TABLE 5 Crossreactivity to Levetiracetam Metabolites High level of2-pyrrolidone-N-butyric acid was tested. % Crossreactivity = 100 ×(“apparent concentration of levetiracetam”/“concentration ofcrossreactant”) Concentration Levetiracetam Tested Result % CrossCompound (μg/ml) (μg/mL) Reactivity Levetiracetam  30.0 29.9 1002-pyrrolidone-N-butyric acid 150.0 0 0 2-pyrrolidone-N-butyric acid406.4 0 0

The preceding merely illustrates the principles of the embodiments ofthe present invention. It will be appreciated that those skilled in theart will be able to devise various arrangements which, although notexplicitly described or shown herein, embody the principles of theinvention and are included within its spirit and scope. Furthermore, allexamples and conditional language recited herein are principallyintended to aid the reader in understanding the principles of theinvention and the concepts contributed by the inventors to furtheringthe art, and are to be construed as being without limitation to suchspecifically recited examples and conditions. Moreover, all statementsherein reciting principles, aspects, and embodiments of the invention aswell as specific examples thereof, are intended to encompass bothstructural and functional equivalents thereof. Additionally, it isintended that such equivalents include both currently known equivalentsand equivalents developed in the future, i.e., any elements developedthat perform the same function, regardless of structure.

1-47. (canceled)
 48. A compound of the formula:

wherein R₁ is —Y—Z; Y is a linking group selected from the groupconsisting of —(CH₂)n-C(O)—, —C(O)(CH₂)n-, —C(O)(CH₂)n-NHC(O)—,—C(O)(CH₂)n-NHC(O)(CH₂)n-, —(CH₂)nSCH₂C(O)—, —(CH₂)n-C(O)NH—(CH₂)n-,—(CH₂)n-NH—C(O)—, and —(CH₂)n-NH—, and n is an integer from 1 to 10; andZ is selected from the group consisting of an immunogenic carrier, afluorophore and an enzyme, and acid salts thereof.
 49. The compound ofclaim 48, wherein Z is the enzyme.
 50. The compound of claim 49, whereinthe enzyme is glucose-6-phosphate dehydrogenase (G6PDH).
 51. Thecompound of claim 49, wherein the enzyme is selected from alkalinephosphatase, β-galactosidase, and horse radish peroxidase.
 52. Thecompound of claim 48, wherein Z is the immunogenic carrier.
 53. Thecompound of claim 52, wherein the immunogenic carrier is BSA or KLH. 54.The compound of claim 52, wherein the immunogenic carrier is selectedfrom the group consisting of hemocyanins, globulins, albumins, andpolysaccharides.
 55. The compound of claim 52, wherein the immunogeniccarrier is selected from the group consisting of a protein, a peptide, aglycoprotein, a saccharide, a nucleic acid and a polynucleotide.
 56. Akit for determining an amount of levetiracetam in a medium, wherein thekit comprises: a) an antibody that binds an epitope present inlevetiracetam and a compound of claim 48; and b) a compound of claim 48,wherein Z is the enzyme.
 57. The kit of claim 56, wherein the enzyme isG6PDH.
 58. A reaction mixture comprising: a) a sample suspected ofcontaining levetiracetam; b) an antibody that binds an epitope presentin levetiracetam and a compound of claim 48; and c) a compound of claim48, wherein Z is the enzyme.
 59. The reaction mixture of claim 58,wherein the enzyme is G6PDH.
 60. A method for detecting levetiracetam,the method comprising: combining in a reaction mixture: (a) a samplesuspected of containing levetiracetam; (b) an antibody that binds anepitope present in levetiracetam and a compound of claim 48; and (c) acompound of claim 48, wherein Z is the fluorophore or the enzyme;allowing one or more of the levetiracetam in the sample and the compoundof claim 48 to bind with the antibody; and measuring the amount of thecompound of claim 48 in the mixture that is bound or unbound to theantibody, whereby the presence of levetiracetam in the sample can bedetected.
 61. The method of claim 60, wherein Z is the enzyme.
 62. Themethod of claim 61, wherein the enzyme is G6PDH.
 63. The method of claim60, wherein Z is the fluorophore.
 64. The method of claim 63, thefluorophore is selected from the group consisting of a naphthalenederivative, an anthracene derivative, a pyrene derivative, a fluoresceinderivative, a rhodamine derivative, and a phycoerythin.